sFRP and peptide motifs that interact with sFRP and methods of their use

ABSTRACT

This disclosure relates to a peptide motif and proteins containing the motif that are capable of binding to secreted Frizzled-related protein family members. Accordingly, the disclosure also includes methods of regulating the interaction of sFRP-1 with proteins containing the motif.

REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 10/466,136,filed Jul. 10, 2003 now U.S. Pat. No. 7,488,710, which is the U.S.National Stage of International Application No. PCT/US02/00869, filedJan. 10, 2002, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.60/260,908, filed Jan. 10, 2001. The entire disclosures of each of theseapplications are hereby expressly incorporated by reference.

FIELD OF THE DISCLOSURE

This disclosure relates to osteoclast differentiation, specifically to apeptide motif and proteins containing the motif that are capable ofbinding to secreted Frizzled-related protein family members.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “Replacement_Sequence_Listing.txt”, created on Jul. 18, 2012, andhaving a size of 26.4 kilobytes, as permitted under 37 CFR 1.821(c). Thematerial in the aforementioned file is hereby incorporated by referencein its entirety.

BACKGROUND

Bone remodeling, a process responsible for the continuous renewal of theadult human skeleton, is carried out by osteoclasts and osteoblasts, twospecialized cell types that originate from hematopoietic and mesenchymalprogenitors of the bone marrow, respectively. A continuous and orderlysupply of these cells is essential for skeletal homeostasis, asincreased or decreased production of osteoclasts or osteoblasts and/orchanges in the rate of their apoptosis are largely responsible for theimbalance between bone resorption and formation that underlies severalsystemic or localized bone diseases.

Enhanced osteoclast activity plays a major role in the pathogenesis ofpostmenopausal osteoporosis, Paget's disease, lytic bone metastases,multiple myeloma, hyperparathyroidism, rheumatoid arthritis,periodontitis, and hypercalcemia of malignancy. These clinical problemsare associated with significant morbidity or mortality, and affect morethan 10 million patients in the United States. However, only a limitednumber of agents that inhibit osteoclast formation or bone resorptionare available and for most their mechanisms of action are unknown.Furthermore, many of these agents have significant side effects thatlimit their utility. Thus, there exists a need for the identificationand characterization of inhibitors of osteoclast formation and boneresorption as part of the continuing search to provide therapeuticbenefits for these patients.

Conversely, decreased osteoclast activity plays a major role in thepathogenesis of osteopetrosis, Albright's osteodystrophy, andachondroplasia, for which there is no specific therapy. Thus, there alsoexists a need for the identification and characterization of treatmentsthat enhance osteoclast formation and bone resorption in order toprovide successful therapies for these patients.

Identification of the mechanisms involved in bone disorders is crucialfor the understanding of bone physiology. While numerous genes and genefamilies (and the polypeptides encoded by them) that participate in theregulation of bone cells have been identified and cloned, theirfunctions have not been clearly delineated due to the complexities ofthe bone formation pathways. A great need exists for the definitiveidentification of targets for the treatment of bone disorders, includingbone resorption disorders such as postmenopausal osteoporosis, Paget'sdisease, lytic bone metastases, multiple myeloma, rheumatoid arthritis,hypercalcemia of malignancy, osteopetrosis, Albright's osteodystrophy,and achondroplasia.

SUMMARY OF THE DISCLOSURE

Disclosed herein are proteins that bind to secreted Frizzled-relatedprotein-1 (-sFRP-1). In one embodiment, the sFRP-1 binding peptide is apurified peptide. In particular examples, the peptide is selected fromthe group consisting of: (a) the amino acid sequence shown in SEQ ID NO:9; (b) at least one conservative amino acid substitution of the aminoacid sequence shown in (a); and (c) an amino acid sequence that sharesat least 80% sequence identity with the sequence shown in (a), whereinthe protein retains the ability to bind to sFRP. In another embodiment,the peptide has a sequence as shown in the formula:[R1]x-R2-R3-R4-R5-R6-R7-R8-[R9]ywherein x and y are integers independently selected from the group 0 or1; R3 is selected from the group Val (V), Ala (A) or conservativesubstitutions therefor; R4 is selected from the group consisting of Asp(D), Ala (A) or conservative substitutions therefor; R5 is selected fromthe group consisting of Gly (G), Ala (A) or conservative substitutionstherefor; R6 is selected from the group consisting of Arg (R) Ala (A) orconservative substitutions therefor; R7 is selected from the groupconsisting of Trp (W), Ala (A) or conservative substitutions therefor.Nucleic acids encoding these peptides are provided, as are vectorscontaining the nucleic acids and host cells transformed with thesevectors. Methods for screening for agents that interfere with or mimicthe interaction of these peptides and sFRP are also disclosed.

In another embodiment, a method is disclosed for enhancing osteoclastdifferentiation. In one specific, non-limiting example the methodincludes administering a therapeutically effective amount of thepurified peptides disclosed herein (or effective fragments, fusions ormimetics) to a subject in order to enhance osteoclast differentiation.

In a further embodiment, a method is provided for inhibiting osteoclastformation in a subject. The method includes administering to the subjecta therapeutically effective amount of sFRP-1 (SEQ ID NO: 3), fragmentsof SEQ ID NO: 3, or fusions or variants of SEQ ID NO: 3, to a subject,wherein the polypeptide binds to a RANKL molecule as set forth asGenBank Accession No. AF013171, GenBank Accession No. AF019047, orGenBank Accession No. AF053712, or another TNF family member.

In yet another embodiment, a method is provided for modulating T cellactivity. In one specific, non-limiting example, the method includesadministering a therapeutically effective amount of the purifiedsFRP-1-binding peptides disclosed herein to a subject in order tomodulate T cell activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the results from A-C2 (SEQ ID NO: 14)/AP(alkaline phosphatase) fusion protein binding to sFRP-1 (SEQ ID NO: 3).Broths from separate bacterial colonies infected with phage expressingthe A-C2/AP chimera were incubated in ELISA wells coated with sFRP-1(and subsequently blocked with BSA) or BSA alone. Each of the broths(identified as I-1, I-2, I-6, I-12, I-13 and I-14) contained APactivity, as measured by reaction with pNPP and color development at 405nm, that bound specifically to the sFRP-1 (SEQ ID NO: 3)-coated wells(white bars) as compared to the wells coated only with BSA (stippledbars). Each sample was tested singly; this is representative of severalexperiments.

FIG. 2 is a graph showing the results from a competitive binding assayof A-C2/AP (SEQ ID NO: 14) and the 12-mer peptides A-C2 (SEQ ID NO: 14)and A-F7 (SEQ ID NO: 12) to either BSA or sFRP-1 (SEQ ID NO: 3). Soluble12-mer peptides, A-C2 (SEQ ID NO: 14) and A-F7 (SEQ ID NO: 12), werepreincubated for 30 minutes at the indicated concentrations withbacterial broth containing A-C2/AP chimera prior to addition to ELISAwells coated with sFRP-1 (A-C2 open squares and A-F7 open diamonds) orBSA (A-C2 open circles and A-F7 open triangles). Samples were tested intriplicate, and results are shown as the mean +/−S.D. This isrepresentative of three experiments.

FIG. 3 is a graph showing the results from binding assays of alaninesubstituted A-C2 (SEQ ID NO: 14)/alkaline phosphatase fusion proteins tosFRP-1 (SEQ ID NO: 3) in an ELISA. The bar graph indicates the mean+/−S.D. of triplicate measurements from a single representativeexperiment. A parallel analysis of these samples in wells coated withmonoclonal antibody to the FLAG epitope indicated that the concentrationof chimera in the different broths was similar.

FIG. 4 is a set of diagrams showing a calorimetric analysis of theinteraction between AC2 peptide and sFRP-1. FIG. 4A is a tracing andplot showing the heat generated when aliquots of AC2 solution were addedto a chamber containing sFRP-1 dissolved in PBS. FIG. 4B is a tracingand plot of the heat generated in the corresponding PBS control. Basedon the amount of heat released, various parameters of the bindingreaction were calculated, including the enthalpy (ΔH) and dissociationconstant, Kd.

FIG. 5 is a graph showing the results from ELISA binding assays usingsoluble RANKL (sRANKL) and sFRP-1 (SEQ ID NO: 3). The open diamondrepresents sRANKL binding to sFRP-1, and the plus symbol “+” representssRANKL binding to bovine serum albumin (BSA).

FIG. 6 is a set of graphs showing that sFRP-1 inhibits osteoclastformation in two different experimental models. FIG. 6A is a graphshowing results from co-culture experiments in which primary osteoblastsand bone marrow were incubated with varying concentrations of sFRP-1.Subsequently, wells were stained to determine the number of TRAP+multinucleated cells (MNC). Results show that as the concentration ofsFRP-1 increases, osteoclast maturation decreases (as is evident bydecrease in TRAP+). The results shown are the mean +/−S.D. ofquadruplicate measurements. FIG. 6B is a graph showing the results fromexperiments in which adult spleen cells were treated with RANKL,macrophage colony stimulating factor (M-CSF), and various concentrationsof sFRP-1 (SEQ ID NO: 3). Subsequently, wells were stained to determinethe number of TRAP+ multinucleated cells (MNC). The data presented isthe mean +/−S.D. of quadruplicate measurements. Results show that as theconcentration of sFRP-1 increases osteoclast maturation decreases.

FIG. 7 is a pair of graphs showing that sFRP-1 antiserum stimulatesosteoclast formation in co-cultures of primary osteoblasts and adultspleen cells. FIG. 7A is a graph showing that sFRP-1 specific antibodybinding to sFRP-1 causes an increase in osteoclast formation. Osteoclastformation, as measured by TRAP+ staining, was assessed in co-cultures ofprimary osteoblasts and adult spleen cells without hormonal supplementsor with suboptimal doses of 1α, 25(OH₂) vitamin D₃ (10⁻¹⁰M) anddexamethasone (10⁻⁹M) in the presence or absence of purifiedimmunoglobulin (˜2 μg/mL) from a rabbit immunized with recombinantsFRP-1. The results are the mean +/−S.D. of mononucleated andmultinucleated TRAP+ cells detected in quadruplicate samples after 7days in culture. FIG. 7B is a graph showing that sFRP-1 specificantibodies bind to sFRP-1 and cause an increase in osteoclast formationin the presence of optimal doses of 1α,25(OH₂) vitamin D₃ (10⁻⁸ M) andprostaglandin E2 (PGE2) 10⁻⁷ M. Osteoclast formation, as measured byTRAP+staining, was assessed in co-cultures of primary osteoblasts andadult spleen cells without hormonal supplements or with optimal doses of1α,25(OH₂) vitamin D₃ (10⁻⁸M) and prostaglandin E2 (PGE2, 10⁻⁷M) in thepresence or absence of purified immunoglobulin (˜1 μg/mL) from a rabbitimmunized with recombinant sFRP-1. The results are the mean +/−S.D. ofmononucleated and multinucleated TRAP+ cells detected in quadruplicatesamples after 7 days in culture.

FIG. 8 is a graph showing that A-C2 peptide stimulates osteoclastformation in co-cultures of osteoblasts and adult spleen cells.Osteoclast formation in response to optimal doses of 1α,25 (OH₂) vitaminD₃ (10⁻⁸M) and PGE2 (10⁻⁷M) was not further enhanced by the concomitantaddition of the netrin homology domain (NHD) domain of sFRP-1 (SEQ IDNO: 13; 5 μg/mL), but it was markedly stimulated by simultaneousincubation with the A-C2 peptide (SEQ ID NO: 14; 5 μg/mL). As a positivecontrol for enhanced osteoclastogenesis, cells were treated withsuboptimal doses of 1α,25(OH₂) vitamin D₃ (10⁻¹⁰M) and PGE2 (10⁻⁹M) inthe absence or presence of sFRP-1 specific antibody 1/500.

FIG. 9 is a graph showing the results from an experiment in which A-C2(SEQ ID NO: 14) was incubated for various time periods with adult spleencells. Group 1 was the control that did not contain A-C2 (SEQ ID NO: 14)peptide. Group 2 was treated with A-C2 (SEQ ID NO: 14) from day 0-3,group 4 was treated with A-C2 (SEQ ID NO: 14) from day 4-7, group 4 wastreated with A-C2 (SEQ ID NO: 14) from day 7-10, and group 5 was treatedwith A-C2 (SEQ ID NO: 14) from day 0-10. All treatment groups receivedRANKL at 50 ng/mL and M-CSF at 25 ng/mL. A-C2 (SEQ ID NO: 14) presenceduring days 0-3 caused an increase in osteoclast production.

FIGS. 10A and 10B are graphs depicting the results from A-C2 (SEQ ID NO:14) incubation with adult spleen cells containing T cells (FIG. 10A) andspleen cells without T cells (FIG. 14B): T cells were immunomagneticallyseparated from the spleen cells. Osteoclast formation was induced byRANKL (50 ng/mL) and M-CSF (25 ng/mL) and assessed by counting TRAP+multinucleated cells after nine days of culture. Osteoclast formationwas measured in adult spleen cell cultures (FIG. 14A) or in culturelacking T cells (FIG. 10B) in the absence (Control +ve) or presence ofA-C2 (5 mg/mL). These cultures do not contain any osteoblasts, thuseffects of A-C2 were restricted to lymphocytic or hematopoietic cells.The bar graphs show the mean +/−S.D. of TRAP+ multinucleated cells fromquadruplicate samples. Controls for this experiment included spleencells [complete (FIG. 10A) or T cell depleted (FIG. 10B)] in the absenceof RANKL and M-CSF (control −ve) and no osteoclasts were produced underthese conditions. As a positive control for the assay system (Control+ve), cultures were treated with RANKL (50 ng/mL) and M-CSF (25 ng/mL),and the effects A-C2 addition is compared to this culture.

FIGS. 11A and 11B are graphs depicting the A-C2 (SEQ ID NO: 14)stimulation of TRAP+, multinucleated cell differentiation in RAW264.7(TIB-71) cell cultures. Group 1 was the positive control that contained50 ng/mL RANKL. Groups 2, 3, and 4, contained 50 ng/mL RANKL and either5 μg/mL, 1 μg/mL, and 0.5 μg/mL of A-C2 (SEQ ID NO: 14) respectively.Stimulation was observed when T cells were added to the cultures (FIG.11A) as compared to when T cells were not added to the cultures (FIG.11B).

FIG. 12 is a graph of the binding avidity of several sFRP-1 deletionmutants for RANKL in ELISA experiments. Wells were coated either withfull-length sFRP-1 or with any one of a set of epitope-tagged sFRP-1deletion mutants (Uren et al., J Biol Chem 275:4374-4382, 2000) or BSAcontrol, and then sequentially incubated with soluble RANKL and reagentsto detect RANKL bound to the wells. The results shown are the mean+/−S.D. of triplicate measurements from a representative experiment.

FIG. 13 is a set of four graphs showing that binding of RANKL tobacterially expressed CRD in ELISA experiments is strong and may havetwo affinities. FIG. 13A is a graph showing the binding of RANKL towells coated with the CRD. Optical density in the wells is a measure ofthe amount of RANKL retained in the wells and is plotted as a functionof the soluble RANKL concentration incubated in the wells. FIG. 13B is aScatchard plot of the RANKL binding data shown in FIG. 13A. The bindingappears to be characterized by more than one affinity. FIG. 13C is areformatting of the Scatchard analysis of FIG. 13B, pertaining to aputative higher-affinity binding site. FIG. 13D is a reformatting of theScatchard analysis of FIG. 13B, pertaining to a putative lower-affinitybinding site.

FIG. 14 is a set of three graphs showing that the bacterially expressedCRD of sFRP-1 inhibits osteoclast formation in a variety of experimentalmodels, including one that is not dependent on RANKL. These were: (1)RAW264.7+TNFα+TGFβ (FIG. 14A; Horwood et al., Journal of Immunology166:4915-4921, 2001; Quinn et al., Journal of Bone and Mineral Research.16, 1787-1794, 2001 (2) the macrophage/monocyte cell line RAW264.7+RANKL(FIG. 14B), and (3) bone marrow cells+RANKL+M-CSF (FIG. 14C). In eachsystem, both RANKL-dependent (FIG. 14B and FIG. 14C) andRANKL-independent (FIG. 14A, TNFα-dependent osteoclast formation), thebacterially expressed CRD mimicked the action of full-length sFRP-1 andwith similar potency.

FIG. 15 is a graph showing that sFRP-1 can inhibit osteoclast formationin RAW264.7 cells treated with a combination of cytokines that includesTNFα, but not RANKL. The effect of sFRP-1 was assessed upon aRANKL-independent method of osteoclast formation using themonocyte/macrophage cell line RAW264.7 (Quinn et al., Journal of Boneand Mineral Research. 16, 1787-1794, 2001) and was compared with that ofosteoprotegerin. TGFα was added during the first three days of cultureto increase osteoclast numbers. sFRP-1 inhibited TNFα-dependentosteoclast formation when present during the first three days ofculture, whilst OPG had no effect suggesting that sFRP-1 was actingindependently of RANKL, through binding to TNFα or through WNTsignaling.

FIG. 16 is a schematic diagram of one possible mechanism of sFRP-1 (SEQID NO: 3)/RANKL binding. Note that the sFRP-1 binding motif in the RANKLsequence is located just downstream from TACE cleavage sites (arrows).TACE is the TNFα converting enzyme, which is known to process RANKL (L.Lum et al., J. Biol. Chem. 274: 13613-13618, 1999). sFRP-1 binding toRANKL could alter the processing of RANKL by TACE, which in turn couldalter RANKL activity.

FIG. 17 is a diagram showing one possible model of sFRP-1's role inosteoclast formation. An osteoclast-supporting cell expressing RANKLinteracts with sFRP-1 (SEQ ID NO: 3) resulting in the inhibition ofosteoclast formation. When the peptide motif (SEQ ID NO: 9) is added tothe solution it binds to sFRP-1 (SEQ ID NO: 3) and promotes osteoclastdifferentiation.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three-letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the cDNA sequence of human sFRP-1.

SEQ ID NO: 2 shows the nucleic acid sequence of the human sFRP-1 openreading frame.

SEQ ID NO: 3 shows the amino acid sequence of human sFRP-1.

SEQ ID NO: 4 shows the amino acid sequence of human sFRP-1-M/H.

SEQ ID NO: 5 shows the amino acid sequence of human sFRP-Δ1-M/H.

SEQ ID NO: 6 shows the amino acid sequence of human sFRP-Δ2-M/H.

SEQ ID NO: 7 shows the amino acid sequence of human sFRP-Δ3-M/H.

SEQ ID NO: 8 shows the amino acid sequence of human sFRP-ΔCRD-M/H.

SEQ ID NO: 9 shows the amino acid sequence of the peptide motif.

SEQ ID NO: 10 shows the peptide motif from ANP receptor A (human).

SEQ ID NO: 11 shows the amino acid sequence of the A-E4 peptide.

SEQ ID NO: 12 shows the amino acid sequence of the A-F7 peptide.

SEQ ID NO: 13 shows the amino acid sequence of the netrin homologydomain of sFRP-1.

SEQ ID NO: 14 shows the amino acid sequence of the A-C2 peptide.

SEQ ID NO: 15-26 show peptides generated for use in alanine scanningexperiments.

SEQ ID NO: 27 shows the amino acid sequence of B-B9.

SEQ ID NO: 28 shows an amino acid sequence found in RANKL that containsa sequence similar to that of SEQ ID NO: 9.

SEQ ID NO: 29 shows an amino acid sequence found in a netrin receptorthat contains a sequence similar to that of SEQ ID NO: 9.

SEQ ID NOS: 30-39 show the nucleic acid sequences of various primers andprobes used in PCR and hybridization experiments.

SEQ ID NO: 40 shows the amino acid sequence of the A-D9 peptide.

DETAILED DESCRIPTION

I. Abbreviations

BSA: bovine serum albumin

CRD: cysteine-rich domain

ELISA: enzyme-linked immunosorbent assay

HSPG: heparin-sulfate proteoglycan

mAb: monoclonal antibody

MDCK: Madin-Darby canine kidney

M/H: Myc-His epitope tags

PAGE: polyacrylamide gel electrophoresis

PBS: phosphate-buffered saline

sFRP: secreted Frizzled-related protein

Wnt: Wnt proteins

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Abnormal: Deviation from normal characteristics. Normal characteristicscan be found in a control, a standard for a population, etc. Forinstance, where the abnormal condition is a disease condition, such asosteoporosis (characterized by a decrease in bone mass), a fewappropriate sources of normal characteristics might include anindividual who is not suffering from the disease (e.g. osteoporosis), apopulation standard of individuals believed not to be suffering from thedisease, etc.

Likewise, abnormal can refer to a condition that is associated with adisease. The term “associated with” includes an increased risk ofdeveloping the disease as well as the disease itself. For instance, acertain abnormality (such as a decrease in the expression of sFRP, whichin turn upregulates osteoclast formation) can be described as beingassociated with the biological condition of osteoporosis (decrease inbone mass); thus, the abnormality is predictive both of an increasedrisk of developing osteoporosis and of the presence of osteoporosis.

Abnormal protein expression, such as abnormal sFRP protein expression,refers to expression of a protein that is in some manner different fromexpression of the protein in a normal (wildtype) situation. Thisincludes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few amino acid residues tothe sequence of the protein; (3) a longer deletion or addition of aminoacid residues, such that an entire protein domain or sub-domain isremoved or added; (4) expression of an increased amount of the protein,compared to a control or standard amount; (5) expression of a decreasedamount of the protein, compared to a control or standard amount; (6)alteration of the subcellular localization or targeting of the protein;(7) alteration of the temporally regulated expression of the protein(such that the protein is expressed when it normally would not be, oralternatively is not expressed when it normally would be); (8)alteration in post translational processing; and (9) alteration of thelocalized (e.g. organ or tissue specific) expression of the protein(such that the protein is not expressed where it would normally beexpressed or is expressed where it normally would not be expressed),each compared to a control or standard.

Controls or standards appropriate for comparison to a sample, for thedetermination of abnormality, include samples believed to be normal aswell as laboratory values, even though possibly arbitrarily set, keepingin mind that such values can vary from laboratory to laboratory.Laboratory standards and values can be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits easy comparison of measured, experimentallydetermined values.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

CRD: A cysteine rich domain that typically is about 120 amino acids inlength and found on the amino terminal half of Fz proteins. In theprototypical sFRP described herein, the CRD comprises sFRP-1 residues38-166. Met (ATG) was added at the N-terminus to facilitate proteinexpression. Typically the Met is cleaved in the bacteria as the proteinis processed. The CRD sequence is shown below:

(amino acids 38-166 of SEQ ID NO: 3)MFQSDIGPYQ SGRFYTKPPQ CVDIPADLRL CHNVGYKKMVLPNLLEHETM AEVKQQASSW.VPLLNKNCHA GTQVFLCSLFAPVCLDRPIY PCRWLCEAVRDSCEPVMQFF GFYWPEMLKC.DKFPEGDVCI

Detectable marker or label: A “detectable marker” or “label” is anymolecule or composition that is detectable by, for instance,spectroscopic, photochemical, biochemical, immunochemical, electrical,optical, or chemical means. Examples of labels, including radioactiveisotopes, enzyme substrates, co-factors, ligands, chemiluminescent orfluorescent agents, haptens, enzymes, colloidal gold particles, coloredlatex particles, and epitope tags, have been disclosed previously andare known to those of ordinary skill (see, for instance, U.S. Pat. Nos.4,275,149; 4,313,734; 4,373,932; and 4,954,452).

Epitope tags are short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been addedto a living organism or to cultured cells. Detection of the taggedmolecule can be achieved using a number of different techniques.Examples of such techniques include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography. Examplesof useful epitope tags include FLAG, T7, HA (hemagglutinin) and myc.

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e. fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forexample Cy3, FITC, and Oregon Green, are characterized by their emissionat wavelengths generally in the range of 515-540λ. Red fluorophores, forexample Texas Red, Cy5 and tetramethylrhodamine, are characterized bytheir emission at wavelengths generally in the range of 590-690λ.

Examples of fluorophores that may be used are provided in U.S. Pat. No.5,866,366, and include for instance:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid(EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide,Brilliant Yellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron .RTM. Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

Other suitable fluorophores include GFP (green fluorescent protein),Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl,naphthofluorescein, 4,7-dichlororhodamine and xanthene and derivativesthereof. Other fluorophores known to those skilled in the art may alsobe used.

Fusion protein: A protein comprising two amino acid sequences that arenot found joined together in nature. The term “sFRP peptide motif fusionprotein” refers to a protein that comprises a first amino acid sequencethat binds sFRP and a second amino acid sequence. The sFRP binding motifand the second amino acid sequence may alternatively be referred to asdomains of the fusion protein. Thus, for example, the present disclosureprovides fusion proteins comprising first and second domains, whereinthe first domain includes a peptide motif that binds sFRP. The linkbetween the first and second domains of the fusion protein is typically,but not necessarily, a peptide linkage.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs (i.e. other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles). Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Linker group or linking group: A linking group is a “chemical arm”between a protein or peptide and a detectable marker. As one of skill inthe art will recognize, to form a chemical arm linker, each of thereactants must contain the necessary groups to link the peptide to thedetectable marker. Representative combinations of such groups are aminowith carboxyl to form amide linkages, or carboxy with hydroxy to formester linkages or amino with alkyl halides to form alkylamino linkages,or thiols with thiols to form disulfides, or thiols with maleimides oralkylhalides to form thioethers. Hydroxyl, carboxyl, amino and otherfunctionalities, where not present may be introduced by known methods.Likewise, as those skilled in the art will recognize, a wide variety oflinking groups may be employed. The structure of the linkage should be astable covalent linkage formed to attach the protein or peptide to thedetectable marker or label. In some cases the linking group may bedesigned to be either hydrophilic or hydrophobic in order to enhance thedesired binding characteristics of the ligand and the receptor. Thecovalent linkages should be stable relative to the solution conditionsunder which the ligand and linking group are subjected. Generallypreferred linking groups will be from 1-20 carbons and 0-10 heteroatoms(NH, O, S) and may be branched or straight chain. Without limiting theforegoing, it should be obvious to one skilled in the art that onlycombinations of atoms that are chemically compatible comprise thelinking group. For example, amide, ester, thioether, thiol ester, keto,hydroxyl, carboxyl, ether groups in combinations with carbon-carbonbonds are acceptable examples of chemically compatible linking groups.

Mimetic: A molecule (such as an organic chemical compound) that mimicsthe activity of a protein, such as sFRP or its fragments, the peptidemotif (such as SEQ ID NO: 9 or SEQ ID NO: 40), or variants or fusionsthereof. Peptidomimetic and organomimetic embodiments are within thescope of this term, whereby the three-dimensional arrangement of thechemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid sidechains in the peptide, resulting in such peptido- andorganomimetics of the peptides having substantial specific inhibitoryactivity or agonist activity. For computer modeling applications, apharmacophore is an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido- andorganomimetics can be designed to fit each pharmacophore with currentcomputer modeling software (using computer assisted drug design orCADD). See Walters, “Computer-Assisted Modeling of Drugs,” in Klegerman& Groves, eds., Pharmaceutical Biotechnology, Interpharm Press: BuffaloGrove, Ill., pp. 165-174, 1993 and Principles of Pharmacology (ed.Munson), chapter 102, 1995, for a description of techniques used incomputer assisted drug design.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length. In several embodiments an oligonucleotide isat least 10, 20, 30, 40, or 50 nucleotides in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Osteoclast: Osteoclasts are large, multinucleate cells that activelyreabsorb bone. Osteoclasts are derived from hematopoietic stem cells andshare phenotypic characteristics with circulating monocytes and tissuemacrophages. They are formed from a population of the circulatingmononuclear cells that are recruited from the blood to the bone surfacewhere they undergo differentiation and fusion to form multinucleatedcells.

Osteopetrosis is a family of diseases characterized by the failure ofthe long bones to be remodeled. The resulting long bones havecartilagenous infiltration towards the center of the bone from thegrowth plate and a poorly remodeled center. While osteoporosis can becaused by too many osteoclasts, osteopetrosis can be caused by nothaving sufficient numbers of these cells.

Loss of ovarian function following menopause often results in aprogressive loss of trabecular bone mass and eventually to osteoporosis.This bone loss is in part due to the increased production ofosteoclasts. This increased production of osteoclasts appears to be dueto the increased elaboration by support cells of osteoclastogeniccytokines such as IL-1, tumor necrosis factor, and IL-6, all of whichare negatively regulated by estrogens.

Osteoclasts are commonly found in degenerative bone diseases at sites ofosteolysis. Osteoclast overproduction is associated with diseases suchas hyperparathyroidism and Paget's disease. Osteoclasts are also seen atsites of inflammatory reactions associated with aseptic loosening oftotal hip prosthesis, rheumatoid arthritis, and periodontitis. Twocytokines produced by inflammatory cells that may have direct effects onosteoclast formation and function are interleukin-1 (IL-1) and tumornecrosis factor (TNF-α).

Peptide motif: An amino acid sequence that binds sFRP-1. Generally, apeptide motif is sequence of two or more peptide-linked amino acids thatprovides a characteristic structure and or function. In one embodiment,a peptide motif can be found in more than one protein or more than oncein a single protein. For example, the peptide motif shown in SEQ ID NO:9 is characterized by its ability to bind to sFRP and modulate sFRPactivity. Without being bound by theory, the three core residues of SEQID NO: 9 (D-G-R) are believed to be important for sFRP-1 binding. Thus,in one embodiment, a peptide motif includes these three amino acids. Inanother embodiment a peptide motif includes the five core amino acids ofSEQ ID NO: 9 (V-D-G-R-W). In addition to the prototypical peptide motifthere are several other examples of motifs (SEQ ID NOS: 9-11, 14-17, and24-26) that bind to sFRP and can be capable of modulating sFRP activity.

While the amino acid sequence of one embodiment of the peptide motifthat binds sFRP-1 is shown in SEQ ID NO: 9, one of skill in the art willappreciate that variations in this amino acid sequence, such as 1, 2, or3 deletions, additions, or substitutions, can be made withoutsubstantially affecting the activities of the peptide motif. Thus, theterm “peptide motif” encompasses both the motif provided in SEQ ID NO:9, and the additional peptide motifs provided in SEQ ID NOS: 10 and 11and 14-26, as well as amino acid sequences that are based on thesesequences but which include one or more sequence variants and fragmentsof these sequences that contain at least 3, 4, 5, or 6 contiguous aminoacids of the peptide motif. Such sequence variants or fragments can alsobe defined in the degree of amino acid sequence identity that they sharewith the amino acid sequence shown in SEQ ID NO: 9. Typically, peptidemotif sequence variants will share at least 80% sequence identity withthe sequences shown in SEQ ID NOS: 9-12 and 14-26. More highly conservedvariants will share at least 90%, at least 95%, or at least 98% sequenceidentity with the sequences shown in SEQ ID NOS: 9-12, 14-17, and 24-26.

The peptide motif is characterized by its ability to bind to sFRP. Thisactivity can be tested using the ELISA assay described below in themethods section. The peptide motifs ability to bind to sFRP and modulatesFRP activity is beneficial in a number of applications, includingclinical applications such as in the treatment of diseases associatedwith abnormal bone remodeling, and more specifically when increasedosteoclast activity is desired.

Peptide tag: A peptide sequence that is attached (for instance throughgenetic engineering) to another peptide or a protein, to provide afunction to the resultant fusion. Peptide tags are usually relativelyshort in comparison to a protein to which they are fused; by way ofexample, peptide tags are four or more amino acids in length, such as 5,6, 7, 8, 9, 10, 15, 20, or or more amino acids. Usually a peptide tagwill be no more than about 100 amino acids in length, and may be no morethan about 75, no more than about 50, no more than about 40, or no morethan about 30.

Peptide tags confer one or more different functions to a fusion protein(thereby “functionalizing” that protein), and such functions can includeantibody binding (an epitope tag), purification, and differentiation(e.g., from a native protein). In addition, a recognition site for aprotease, for which a binding antibody is known, can be used as aspecifically cleavable epitope tag. The use of such a cleavable tag canprovide selective cleavage and activation of a protein (e.g., byreplacing the cleavage site in TGF-β1 with that for pro-caspase 3.

Detection of the tagged molecule can be achieved using a number ofdifferent techniques. These include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography.

Epitope tags add a known epitope (antibody binding site) on the subjectprotein, providing binding of a known and often high-affinity antibody,and thereby allowing one to specifically identify and track the taggedprotein that has been added to a living organism or to cultured cells.Examples of epitope tags include the myc, T7, GST, GFP, HA(hemagglutinin) and FLAG tags. The first four examples are epitopesderived from existing molecules. In contrast, FLAG is a syntheticepitope tag designed for high antigenicity (see, e.g., U.S. Pat. Nos.4,703,004 and 4,851,341).

Purification tags are used to permit easy purification of the taggedprotein, such as by affinity chromatography. A well-known purificationtag is the hexa-histidine (6×His) tag, literally a sequence of sixhistidine residues. The 6×His protein purification system is availablecommercially from QIAGEN (Valencia, Calif.), under the name ofQIAexpress®.

A single tag peptide can serve more than one purpose; any attached tag,for instance, will increase the molecular weight of the fusion proteinand thereby permit differentiation between the tagged and nativeproteins. Antibodies specific for an “epitope tag” can be used toconstruct an immunoaffinity column, thus permitting an epitope tag to beused for purification of the tagged protein. Likewise, in some instancesmonoclonal antibodies specific for a purification tag are available(e.g. anti-6×His peptide monoclonal antibodies, which are availablethrough QIAGEN or CLONTECH, Palo Alto, Calif.).

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g. powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A nucleic acid sequence including at least two nucleicacid residues.

Polypeptide: A protein fragment including at least two amino acidresidues.

Protein Fragment: An amino acid sequence that contains fewer amino acidresidues than are found in a naturally occurring protein and includingat least two amino acid residues. For example, if a naturally occurringprotein, i.e. a protein expressed from a gene, is 300 amino acidresidues long, a polypeptide derived from the protein could have 299amino acid residues or less. In particular examples, the polypeptidecould have less than 200, 175, 150, 125, 100, 75, 50, or 25 amino acidresidues.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified protein orpeptide preparation is one in which the protein or peptide is more purethan the protein or peptide in its natural environment within a cell.Such proteins or peptides may be produced, for example, by standardpurification techniques, or by recombinant expression. In someembodiments, a preparation of a protein or peptide is purified such thatthe protein or peptide represents at least 50%, for example, or at least70%, of the total protein content of the preparation.

RANK and RANKL: The receptor activator of NF-κB (RANK) is a member ofthe tumor necrosis factor (TNF) receptor superfamily. The ligand,receptor activator of NF-KB ligand (RANKL), is a member of the TNFsuperfamily, and has been characterized in multiple settings andvariously termed Osteoclast Differentiation Factor (ODF), Tumor NecrosisFactor-Related Activation-Induced Cytokine (TRANCE) and OsteoprotegerinLigand (OPGL). RANK is a Type I transmembrane protein having 616 aminoacid residues that interacts with TNF-receptor associated factor 3(TRAF3). Triggering of RANK by over-expression, co-expression of RANKand membrane bound RANK ligand (RANKL), or the addition of soluble RANKLor agonistic antibodies to RANK, results in the upregulation of thetranscription factor NF-κβ, a ubiquitous transcription factor that ismost extensively utilized in cells of the immune system (U.S. Pat. No.6,017,729).

RANK is expressed on osteoclast precursors and mature osteoclasts. RANKLproduced by osteoblasts stimulates the formation and activity ofosteoclasts, which facilitates normal bone development and remodeling.Gene targeting of either RANKL or RANK results in osteopetrosis(increased bone mass), as well as severe defects in lymph nodeformation. Osteoprotegerin (OPG) is a soluble factor that also belongsto the TNF receptor family. OPG binds to RANKL, and inhibits theformation of functional multinucleate osteoclasts in vitro.Overexpression of OPG in transgenic mice causes severe osteopetrosis,with a loss of marrow cavities and profound depletion of osteoclasts.The same effects were observed upon administration of OPG in normalmice. These effects were all attributable to OPG's binding to RANKL,which prevented ligand binding and activation of RANK. Alternatively,expression of RANKL by T cells in the joints of subjects afflicted withrheumatoid arthritis is thought to contribute to the heightenedosteoclast activity and bone loss characteristic of this disorder.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g. by genetic engineering techniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of sFRP (the prototypical member of which is shownin SEQ ID NO: 1), or the peptide motif that binds sFRP (for example SEQID NO:9), disclosed herein, will possess a relatively high degree ofsequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higginsand Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119-129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Variants of sFRP, sFRP fragments, or the peptide motif that binds sFRP,are typically characterized by possession of at least 50% sequenceidentity counted over the full length alignment with the amino acidsequence of sFRP, sFRP fragments or the peptide motif (for example SEQID NO: 9) using the NCBI Blast 2.0, gapped blastp set to defaultparameters. For comparisons of amino acid sequences of greater thanabout 30 amino acids, the Blast 2 sequences function is employed usingthe default BLOSUM62 matrix set to default parameters, (gap existencecost of 11, and a per residue gap cost of 1). When aligning shortpeptides (fewer than around 30 amino acids), the alignment should beperformed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 90%, or at least 95%, or 98% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs and variants will typically possess at least75% sequence identity over short windows of 10-20 amino acids, and canpossess sequence identities of at least 85% or at least 90%, 95%, or 98%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe website that is maintained by the National Center for BiotechnologyInformation in Bethesda, Md. One of skill in the art will appreciatethat these sequence identity ranges are provided for guidance only; itis entirely possible that strongly significant homologs could beobtained that fall outside of the ranges provided.

sFRP: Secreted Frizzled-related protein (sFRP) is a secreted proteinthat consists of approximately 300 amino acids, including a CRD that istypically between 30% and 50% identical to the (cysteine-rich domain)CRD of the Fz protein family members. There are several different sFRPproteins and the nucleic acid sequence of the prototypical member,sFRP-1, is provided in SEQ ID NO: 1. The nucleic acid and amino acidsequences of other members of the sFRP family can be found at theNational Center for Biotechnology Website, for example GenBank AccessionNo. AF218056 (Gallus gallus FRP-2), GenBank Accession No AV354083 (Musmusculus-FRP-1), GenBank Accession No AV304328 (Mus musculus s-FRP-2),GenBank Accession No U24163 (homo sapiens sFRP-3/FrzB) and GenBankAccession No AI587049 (Homo sapiens sFRP-1). The open reading frame ofthe prototypical sFRP is shown in SEQ ID NO: 2, while the sequence ofthe protein is shown in SEQ ID NO: 3. As disclosed herein, sFRP binds toRANKL and inhibits osteoclast formation.

sFRP-1 binding activity and its ability to modulate osteoclast formationcan be assayed using the ELISA and osteoclastogenesis bioassay methodsdescribed herein. The ability of sFRP-1 protein, or a fragment thereof,to perform these activities is beneficial in a number of applications,including clinical applications such as in the treatment of diseasesassociated with abnormal bone remodeling.

While the amino acid sequence of the prototypical sFRP is shown in SEQID NO: 3, one of skill in the art will appreciate that variations inthis amino acid sequence, such as 1, 2, 5, 10, 20, 30, 40, or 50,deletions, additions, or substitutions (including conservative aminoacid substitutions), can be made without substantially affecting theactivities of the protein (or fragments of the protein) discussed above.Thus, the term “sFRP” fragments encompasses both the proteins having theamino acid sequences shown in SEQ ID NOs: 4-8, as well as amino acidsequences that are based on these sequences but which include one ormore sequence variants. Such sequence variants can also be defined inthe degree of amino acid sequence identity that they share with theamino acid sequence shown in SEQ ID NOs: 4-8. Typically, sFRP sequencevariants will share at least 80% sequence identity with the sequencesshown in SEQ ID NOs: 4-8. More highly conserved variants will share atleast 90%, at least 95%, or at least 98% sequence identity with thesequences shown in SEQ ID NOs: 4-8. In addition to sharing sequenceidentity with the prototypical sFRP protein sequence, such sequencevariants possess the ability to bind to TNF family members such asRANKL.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease.

TNF family of proteins: The Tumor Necrosis (TNF) family of proteinscontains both membrane bound ligands and soluble proteins. Some familymembers, such as TNF and RANKL, are active in both membrane-anchored andsoluble forms, the latter being enzymatically released into solution,notably by TACE (TNF alpha converting enzyme) (J. Hardy, Proc. Natl.Acad. Sci. U.S.A. 94:2095-2097, 1997; J. D. Buxbaum et al., Proc. Natl.Acad. Sci. U.S.A. 89: 10075-10078, 1992). The primary area of homologyamong TNF family members is a stretch of 150 amino acid residues in thecarboxy-terminus that is situated in the extracellular space. Thisdomain is responsible for binding to cognate members of the TNF receptorfamily. This family of receptor proteins is characterized by fourdomains with regularly spaced cysteine residues: each has a singletransmembrane domain and binds either TNFα or TNFβ. Members of thefamily include, for example, TNFRI, TNFRII, Fas, CD30, and CD30.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector can also include one or more selectable markergenes and other genetic elements known in the art. A vector can alsoinclude a sequence encoding an amino acid motif that facilitates theisolation of the desired protein product such as a sequence encodingmaltose binding protein, c-myc, or GST.

WNT: One group of genes and the proteins encoded by them that play animportant role in regulating cellular development is the Wnt family ofglycoproteins. Wnt proteins are a family of growth factors consisting ofmore than a dozen structurally related molecules and are involved in theregulation of fundamental biological processes, like apoptosis,embryogenesis, organogenesis, morphogenesis and turnorigenesis. Thesepolypeptides are multipotent factors and have similar biologicalactivities to other secretory proteins like transforming growth factor(TGF)-β, fibroblast growth factors (FGFs), nerve growth factor (NGF),and bone morphogenetic proteins (BMPs).

A member of the Wnt growth factor family is preferentially expressed inbone tissue and in bone-derived cells, and appears to be involved inmaintaining the mature osteoblast (bone-forming cell) phenotype.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. “Comprising” means“including.” The singular terms “a”, “an”, and “the” include pluralreferents unless context clearly indicates otherwise. Hence, “comprisingA and B” means “including A and B” without excluding other elements.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Description of Several Embodiments

As disclosed herein, a peptide motif has been demonstrated to bind tosFRP-1 (SEQ ID NO: 3) and inhibit the ability of sFRP-1 to down regulatethe formation of osteoclasts. In one embodiment, the peptide motif hasthe formula:[R1]_(x)-R2-R3-R4-R5-R6-R7-R8-[R9]_(y)

wherein x and y are integers independently selected from the group 0 or1; R1, R2, R8, and R9 are any amino acid residue; R3 is selected fromthe group Val (V), Ala (A) or conservative

substitutions therefore; R4 is selected from the group consisting of Asp(D), Ala (A) or conservative substitutions therefore; R5 is selectedfrom the group consisting of Gly (G), Ala (A) or conservativesubstitutions therefore; R6 is selected from the group consisting of Arg(R) Ala (A) or conservative substitutions therefore; R7 is selected fromthe group consisting of Trp (W), Ala (A) or conservative substitutionstherefore; and wherein the peptide retains the ability to bind to asFRP.

In one embodiment, R1 is selected from the group consisting of (a)Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr (AGT), (c) Gln-Ala-Thr (QAT), and (d)Gln-Gly-Ala (QGA). In another embodiment, R2 is selected from the groupLeu, Val, Ala (L, V, A) or a conservative substitution therefor. In afurther embodiment, R8 is selected from the group consisting of Leu orVal (L or V) or a conservative substitution therefor. In yet anotherembodiment, R9 is selected from the group consisting of (a) Gln (Q), (b)Gln-Gly-Glu (QGE), (c) Gln-Leu (QL), (d) Ala-Leu (AL), (e) Gln-Ala (QA)and (f) Thr-Asn-Pro-His-His (TNPHH) (SEQ ID NO: 41).

In a specific, non-limiting example, R3 is V, R4 is D, R5 is R, R6 is G,and R7 is W. In another specific, non-limiting example, R1 is selectedfrom the group consisting of (a) Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr(AGT), (c) Gln-Ala-Thr (QAT), and (d) Gln-Gly-Ala (QGA); R2 is selectedfrom the group Leu, Val, Ala (L, V, A) or a conservative substitutiontherefor; R8 is selected from the group consisting of Leu or Val (L orV) or a conservative substitution therefor; R9 is selected from thegroup consisting of (a) Gln (Q), (b) Gln-Gly-Glu (QGE), (c) Gln-Leu(QL), (d) Ala-Leu (AL), (e) Gln-Ala (QA) and (f) Thr-Asn-Pro-His-His(TNPHH) (SEQ ID NO: 41); and R3 is V, R4 is D, R5 is R, R6 is G, and R7is W. In yet another non-limiting example R3 is V, R4 is D, R5 is R, R6is G, and R7 is W and R1 is selected from the group consisting of (a)Gln-Gly-Thr (QGT), (b) Ala-Gly-Thr (AGT), (c) Gln-Ala-Thr (QAT), and (d)Gln-Gly-Ala (QGA). In a further non-limiting example R3 is V, R4 is D,R5 is R, R6 is G, and R7 is W R2 is selected from the group Leu, Val,Ala (L, V, A) or conservative substitutions therefor. In anotherspecific, non-limiting example, R3 is V, R4 is D, R5 is R, R6 is G, andR7 is W and R8 is selected from the group consisting of Leu, Val, Ala(L, V, A) and conservative substitutions therefor. In another specific,non-limiting example, R3 is V, R4 is D, R5 is R, R6 is G, and R7 is Wand R9 is selected from the group consisting of (a) Gln (Q), (b)Gln-Gly-Glu (QGE), (c) Gln-Leu (QL), (d) Ala-Leu (AL), (e) Gln-Ala (QA)and (f) Thr-Asn-Pro-His-His (TNPHH) SEQ ID NO: 41). One specific,non-limiting example of a peptide motif is SEQ ID NO: 9.

In one embodiment, the sFRP binding peptide is less than 30 amino acidsin length. In another embodiment, the peptide is less than 20 aminoacids in length. In a further embodiment, the peptide is less than 10amino acids in length.

The identification of a peptide motif that binds sFRP (for example, SEQID NO: 9) has also allowed other proteins to be identified, which arecharacterized by the presence of a sequence resembling the peptide motifin their amino acid sequences, and by their ability to bind to sFRPfamily members. These peptides interfere with sFRP activity, for examplebinding RANKL or a TNF family member, or osteoclastogenesis stimulatingactivity. Accordingly, the disclosure provides methods of controllingbone remodeling. The peptide motif disclosed herein (for example, SEQ IDNO:9) can be used to bind to sFRP-1 and effectively upregulateosteoclast differentiation. Increased osteoclast production is desirablefor the treatment of disorders where there is too much bone formation(for example, achondroplasia, Albright's osteodystrophy, andosteopetrosis). Conversely, the disclosure also provides methods ofproviding sFRP to increase bone mass (see FIG. 17). An increase in bonemass is desirable for the treatment of disorders such as postmenopausalosteoporosis, Paget's disease, lytic bone metastases, multiple myeloma,hyperparathyroidism, rheumatoid arthritis, periodontitis, andhypercalcemia of malignancy.

Some embodiments of the disclosure provide isolated polypeptides,including the amino acid sequence shown in SEQ ID NO: 9; conservativeamino acid substitutions of the amino acid sequence shown in SEQ ID NO:9; and amino acid sequences that share at least 80% sequence identitywith the sequence shown in SEQ ID NO: 9. These polypeptides are capableof binding sFRP-1 (SEQ ID NO: 3) and interfering with sFRP activity, forexample osteoclastogenesis activity. Examples of such polypeptides areprovided in SEQ ID NOS: 10-12 and 14-29.

The disclosure also provides nucleic acid sequences that encode thepeptide motif that binds sFRP and the variants of the peptide motif thatbinds sFRP that are described in the paragraph above. These nucleic acidsequences can be placed in vectors, and the vectors can be used totransform host cells. The transformed host cells are subsequently usefulfor, among other things, producing the above-described polypeptides.

As mentioned above, the disclosure provides methods of enhancingosteoclast differentiation in a subject. These methods include providingan effective amount of a peptide that includes the motif (such as SEQ IDNO: 9), or variants, or fragments thereof to increase osteoclastdifferentiation. Such methods are useful for treating subjects suspectedof having abnormal bone remodeling (e.g. achondroplasia, Albright'sosteodystrophy, or osteopetrosis).

The disclosure also provides methods of inhibiting osteoclast formationin a subject. These methods include administering sFRP-1 (SEQ ID NO: 3),variants of sFRP-1 (SEQ ID NO: 3), or fusions, or fragments of sFRP-1(SEQ ID NO: 3). Administering these peptides includes administration andexpression of nucleic acids that encode the peptides. The administeredproteins or peptides are characterized by their ability to bind toRANKL, for example, human RANKL termed “TRANCE” (AF013171), human RANKL(AF019047), and human RANKL termed “OPGL” (AF053712) and inhibitosteoclast formation. The inhibition of osteoclast formation will beuseful for treating osteopathic disorders such as postmenopausalosteoporosis, Paget's disease, lytic bone metastases, multiple myeloma,hyperparathyroidism, rheumatoid arthritis, periodontitis, andhypercalcemia of malignancy.

The peptide motif that binds sFRP, and fragments and variants thereof,are also useful for modulating T-cell activity. Accordingly, thedisclosure provides methods of modulating T-cell activity. These methodsinclude providing an effective amount of the peptide motif that bindssFRP (such as SEQ ID NO:9), or fragments and variants thereof,sufficient to change T-cell interaction with dendritic cells orosteoclast progenitor cells. Examples of changes in the interactionbetween the T-cell and the dendritic cell include an increase indendritic cell survival, and T cell proliferation, in a mixed lymphocytereaction, as described for RANKL/RANK signaling (D. M. Anderson et al.,Nature 390:175-179, 1997; and B. R. Wong et al., J. Exp. Med.186:2075-2080, 1997). Modulating T-cell activity is desirable insubjects suspected of having, for example, toxic shock, sepsis,graft-versus-host reactions, or acute inflammatory reactions.

The disclosure also provides methods of screening for sFRP proteins, andfragments, and variants thereof, that bind to members of the TNF familyof proteins. These methods include contacting an sFRP protein with atleast one TNF family member, and detecting TNF family member binding tothe sFRP protein. Members of the TNF family that are of particularinterest include RANKL, Apo2/TRAIL, FasL, CD40L, CD27L, CD30L,Apo3L/TWEAK, TNF and LT-alpha (S. J. Baker and E. P. Reddy, Oncogene 17:3261-3270, 1998). Members of the sFRP family that are of particularinterest include sFRP-1 (SEQ ID NO: 3), sFRP-2 (GenBank Accession No.MMU88567, incorporated herein by reference), sFRP-3 (GenBank AccessionNo. MMU88568, incorporated herein by reference), sFRP-4 (GenBankAccession No. AF012891, incorporated herein by reference), and sFRP-5(GenBank Accession No. AF117758, incorporated herein by reference).

The disclosure also provides the purified peptide shown in SEQ ID NO:14. This peptide is useful for stimulating osteoclast differentiation invitro and in vivo. When the peptide is used in vivo it can beadministered to subjects to increase osteoclast differentiation.

IV. Expression and Purification of sFRP, Fragments, Fusions, andVariants Thereof, as Well as the Peptide Motif

sFRP fragments and variants thereof can be purified from MDCK cells(ATCC NO. CCL-34) transfected with sFRP encoding vectors as describedbelow. sFRP fragments and variants thereof can also be purified from atissue source using conventional biochemical techniques, or producedrecombinantly in either prokaryotic or eukaryotic cells using methodswell-known in the art (for example, those described in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).The recombinant expression of sFRP fragments is described in (Uren etal., J. Biol. Chem. 275:4374-4382, 2000). Furthermore, the nucleic acidsequences encoding sFRP family members are available on GenBank, andinclude the cDNA sequence shown in SEQ ID NO: 1.

Recombinant sFRP fragments, fusions, and variants thereof, as well asthe binding motif (SEQ ID NO: 9) and variants thereof, can be obtainedusing commercial systems designed for optimal expression andpurification of fusion proteins. Such fusion proteins typically includea protein tag that facilitates purification. Examples of such systemsinclude: the pMAL protein fusion and purification system (New EnglandBiolabs, Inc., Beverly, Mass.); the GST gene fusion system (AmershamPharmacia Biotech, Inc., Piscataway, N.J.); and the pTrcHis expressionvector system (Invitrogen, Carlsbad, Calif.). For example, the pMALexpression system utilizes a vector that adds a maltose binding proteinto the expressed protein. The fusion protein is expressed in E. coli.,and the fusion protein is purified from a crude cell extract using anamylose column. If necessary, the maltose binding protein domain can becleaved from the fusion protein by treatment with a suitable protease,such as Factor Xa. The maltose-binding fragment can then be removed fromthe preparation by passage over a second amylose column. Eukaryoticexpression systems can also be employed, including Pichia, tobacco andBaculovirus expression systems, such as those available commerciallyfrom Invitrogen.

For each of these systems, the entire sFRP protein, variants andfragments thereof or the peptide binding motif can be produced byligating the open reading frame (ORF) of the desired sequence into thevector. To ensure effective expression, the ORF must be operably linkedto the vector, i.e. must be joined such that the reading frame of theORF is aligned with the reading frame of the protein tag. Wherefragments of sFRP are to be expressed, an ORF encoding the desiredfragment can be amplified by polymerase chain reaction (PCR) from thesFRP cDNA, cloned, purified and then ligated into the expression vector.Alternatively, the amplified fragment can be ligated directly into theexpression vector. It can also be possible, depending on theavailability of suitable restriction sites in the sFRP cDNA, to obtainthe desired fragment by appropriate restriction endonuclease digestion,such that it can be directly cloned into the expression vector.

Purification of the expressed protein can be achieved either using thepurification regimen appropriate for the expression tag (if a commercialexpression/purification system is used), or conventional affinitychromatography using antibodies, preferably monoclonal antibodies, thatrecognize the appropriate regions of sFRP can be employed orchromatography procedures established for sFRPs.

Where sFRP fragments or protein fragments containing the peptide motif(for example, SEQ ID NO: 9) are to be used, such fragments alternativelycan be generated through digestion of a full-length protein with variousproteases. The fragments can then be separated based on their uniquesize, charge or other characteristics. Such fragments can also besynthetically generated through the use of known peptide synthesismethods.

V. Methods of Enhancing or Inhibiting Osteoclast Formation

The peptide motif that binds sFRP can be used to enhance osteoclastdifferentiation. Osteoclasts are large, multinucleate cells thatactively reabsorb bone, are derived from hematopoietic stem cells, andshare phenotypic characteristics with circulating monocytes and tissuemacrophages. They are formed from a population of the circulatingmononuclear cells that are recruited from the blood to the bone surface,where they undergo differentiation and fusion to form multinucleatedcells.

Osteopetrosis is a family of diseases characterized by the failure ofthe long bones to be remodeled. The resulting long bones havecartilagenous infiltration towards the center of the bone from thegrowth plate and a poorly remodeled center. While osteoporosis can becaused by osteoclasts that are too numerous or too active, osteopetrosiscan be caused by not having sufficient numbers of these cells, or bytheir inadequate activity. Thus, enhancement of osteoclastdifferentiation is desirable in subjects with abnormal bone remodeling,such as achondroplasia and osteopetrosis. Methods of administration ofthese FRP-1 to inhibit osteoclast differentiation in a subject aredescribed below.

Conversely, sFRP-1 can be used to inhibit osteoclast formation. Loss ofovarian function following menopause often results in a progressive lossof trabecular bone mass and eventually to osteoporosis. This bone lossis in part due to the increased production of osteoclasts. Thisincreased production of osteoclasts appears to be due to the increasedelaboration by support cells of osteoclastogenic cytokines such as IL-1,tumor necrosis factor, and IL-6, all of which are negatively regulatedby estrogens.

Osteoclasts are also implicated in degenerative bone diseases at sitesof osteolysis. Likewise, osteoclast overproduction is associated withdiseases such as hyperparathyroidism and Paget's disease. Osteoclastsare also seen at sites of inflammatory reactions associated with asepticloosening of total hip prosthesis, rheumatoid arthritis, andperiodontitis. Two cytokines produced by inflammatory cells that mayhave direct effects on osteoclast formation and function areinterleukin-1 (IL-1) and tumor necrosis factor (TNF-α). Thus, inhibitionof osteoclast formation is desirable in subjects with bone disorderscharacterized by unwanted bone resorption.

In view of sFRP-1's ability to inhibit osteoclastogenesis, sFRP-1 canhave clinical utility in conditions where excessive osteoclast activityhas pathological consequences. Osteoporosis and rheumatoid arthritis areexamples of conditions that are particularly good targets for sFRP-1therapy because soluble RANKL from T cells is thought to have animportant role in the bone loss associated with these diseases. Methodsof administration of sFRP-1 to inhibit osteoclast formation in a subjectare disclosed herein.

Disorders of calcium homeostasis can also be affected by osteoclastactivity. For example, osteoclasts are able to mobilize calcium frombone to affect hypocalcemic states. Alternatively, inhibition ofosteoclasts can help minimize mobilization of in hypercalcemic states.Hence, modulation of osteoclast activity can be used as a therapeuticintervention to treat hypocalcemia and hypercalcemia.

VI. Methods of Modulating T-cell Activity in a Subject

The sFRP-binding peptides described herein are effective for treatmentof conditions or diseases that involve the immune system, for instanceconditions (including clinical treatments) that inhibit (or suppress)the immune system. General information about the therapeutic use ofimmunomodulatory compounds is well known, and can be found for instancein U.S. Pat. Nos. 5,632,983; 5,726,156; and 5,861,483.

The peptide motif disclosed herein is of use in modulating antigenpresentation. T-cells produce RANKL, and dendritic cells express RANK.Thus, in order to increase an immune response, T cells can be exposed toa polypeptide including the peptide motif, for example administration ofthe polypeptide to a subject. The administration of the polypeptideresults in an increase in RANKL, and subsequently the binding of RANK toRANKL on T cells. Thus, in the presence of an antigen, administration ofa polypeptide including the sFRP binding peptide disclosed hereinresults in increased antigen presentation, and a correspondingupregulation of an immune response against the antigen. Immunedeficiencies (e.g., deficiencies of one or more type of immune cells, orof one or more immunological factors) associated with immune deficiencydiseases, immune suppressive medical treatment, acute and/or chronicinfection, and aging can be treated using the methods and compositionsdescribed herein. A general overview of immunosuppressive conditions anddiseases can be found in Harrisons “Principles of Internal Medicine,”14^(th) Edition, McGraw-Hill, 1998, and particularly in chapter 86(Principles of Cancer Therapy), chapter 88 (Melanoma and other SkinCancers), chapter 307 (Primary Immune Deficiency Diseases), and chapter308 (Human Immunodeficiency Virus Diseases). In one embodiment apolypeptide including the sFRP binding peptide motif is administered toan immunosuppressed subject, such as a subject receivingimmunosuppressive medical treatment, a subject with an age-linkedimmunodeficiency, or a subject that is infected with a humanimmunodeficiency virus. In another embodiment, the peptides disclosedherein are utilized to activate the immune system against variousdiseases, both chronic and acute. Subject infections include bacterialand viral infections, as well as infestations caused by eukaryoticpathogens and parasites.

More particularly, immunostimulatory sFRP-1-binding peptide-treatmentcan be used in the treatment of HIV disease.

VII. Methods of Regulating Intraocular Pressure and Treating Glaucoma

In addition to the peptide motif s impact on sFRP-1/RANKL binding,compositions containing the peptide motif (such as a compositionincluding a peptide as set forth as SEQ ID NO: 9) also have utility indisrupting the interaction of sFRP-1 with other proteins. For instance,sFRP-1 binding to the ANP receptor A can regulate the release of sodiumand fluid in the kidney and eye. It has been demonstrated that therelevant components of natriuretic peptide system are functionallyexpressed in the human eye where they are believed to serve asmodulators of intraocular pressure (J. Ortego and M. Coca-Prados,Biochem. Biophys. Res. Commun. 258: 21-28, 1999). In the eye, sFRP-1 orits binding peptide can have an important impact on the release of fluidinto the eye with resultant changes in the intraocular pressure. In oneembodiment, a polypeptide that includes a peptide motif that binds sFRP,such as a polypeptide including SEQ ID NO:9, is administered to asubject to decrease intraocular pressure. In one specific non-limitingexample, the polypeptide including the peptide motif that binds sFRP isadministered to decrease intraocular pressure in a subject with glaucoma(see Johnson and R. C. Tschumper, Invest. Ophthalmol. Vis. Sci. 28:945-953, 1987). The peptide can be administered intraocularly (forexample in a sustained release intraocular implant). Alternatively, thepolypeptide may be administered systemically, in a therapeuticallyeffective amount sufficient to inhibit production of aqueous humor inthe anterior chamber of the eye.

VIII. Screening Assays for Detecting sFRP Modulation of TNF-ligandFamily Members

The peptide motif that binds sFRP can be used in screening for theidentification of proteins and other compounds that bind to, orotherwise directly interact with sFRP or fragments thereof, such as amimetic. The proteins include members of the TNF family of proteins suchas, RANKL, TRAIL, FasL, CD40L, CD27L, CD30L, and NGF. In one embodiment,a cell lysate or tissue homogenate can be screened for proteins or othercompounds that disrupt sFRP/TNF or sFRP/peptide motif binding.Alternatively, any of a variety of exogenous compounds, both naturallyoccurring and/or synthetic (e.g. libraries of small molecules orpeptides), can be screened for the ability to disrupt sFRP/TNF orsFRP/peptide motif binding (such as the ability to disrupt binding of apeptide having a sequence as set forth as SEQ ID NO: 9 with TNF orRANKL). Small molecules are particularly preferred in this contextbecause they are more readily absorbed after oral administration, havefewer potential antigenic determinants, and/or are more likely to crossthe blood brain barrier than larger molecules such as nucleic acids orproteins.

Furthermore, the identification of deletion mutants (i.e. the fragmentsof sFRP shown in the sequence listing) that are significantly smallerthan full length sFRP but yet maintain the ability to bind to andregulate TNF proteins provides “lead compounds” for the design anddevelopment of new pharmaceuticals. Similarly, a polypeptide including apeptide motif that binds sFRP can serve as a “lead compound.” Forexample, as is well known in the art, sequential modification of smallmolecules (e.g. amino acid residue replacement with peptides; functionalgroup replacement with peptide or non-peptide compounds) is a standardapproach in the pharmaceutical industry for the development of newpharmaceuticals. Such development generally proceeds from a “leadcompound” which is shown to have at least some of the activity (e.g.modulates osteoclastogenesis) of the desired pharmaceutical. Inparticular, when one or more compounds having at least some activity ofinterest are identified, structural comparison of the molecules cangreatly inform the skilled practitioner by suggesting portions of thelead compounds that should be conserved, and portions that can be variedin the design of new candidate compounds. Thus, the present disclosurealso provides potential lead compounds as well as means of identifyingsuch lead compounds that can be modified sequentially to produce newcandidate compounds for use in the treatment of diseases associated withabnormal osteoclast activity, i.e. arthritis. These new compounds thencan be tested both for TNF receptor binding (in the case of leadcompounds developed from sFRP) or sFRP binding (in the case of leadcompounds developed from the peptide motifs disclosed herein) and forbiological efficacy (e.g. in the osteoclastogenesis assays describedherein). This procedure can be iterated until compounds having thedesired therapeutic activity and/or efficacy are identified.

The effect of agents that disrupt sFRP/peptide motif binding can bemonitored using the osteoclast differentiation assays described below.Agents that disrupt sFRP binding and enhance osteoclastogenesis areuseful for treating conditions associated with increased bone mass andagents that are found to enhance sFRP/TNF binding are useful fortreating diseases associated with decreased bone mass (e.g. see FIG.17). Methods of detecting such binding include the ELISA assaysdescribed below, as well as other methods that involve monitoringchanges in fluorescence, molecular weight, or the concentration ofeither sFRP, or proteins containing the peptide motif that binds sFRPeither in a soluble phase or in a substrate-bound phase. In oneembodiment, the peptide motif has a sequence as set forth as SEQ IDNO:9.

Once identified by the methods described above, the candidate compoundscan then be produced in quantities sufficient for pharmaceuticaladministration or testing (e.g. μg or mg or greater quantities), andformulated in a pharmaceutically acceptable carrier (see, e.g.Remington's Pharmaceutical Sciences, Gennaro, A., ed., Mack Pub., 1990).These candidate compounds can then be administered to the transformedcells of the disclosure, to the transgenic animal models of thedisclosure, to cell lines derived from the animal models or from humanpatients.

The proteins or other compounds identified by these methods can bepurified and characterized by any of the standard methods known in theart. Proteins can, for example, be purified and separated usingelectrophoretic (e.g. SDS-PAGE, 2D PAGE) or chromatographic (e.g. HPLC)techniques and can then be microsequenced. For proteins with a blockedN-terminus, cleavage (e.g. by CNBr and/or trypsin) of the particularbinding protein is used to release peptide fragments. Furtherpurification/characterization by HPLC and microsequencing and/or massspectrometry by conventional methods provides internal sequence data onsuch blocked proteins. For non-protein compounds, standard organicchemical analysis techniques (e.g. IR, NMR and mass spectrometry;functional group analysis; X-ray crystallography) can be employed todetermine their structure and identity.

Methods for screening cellular lysates, tissue homogenates, or smallmolecule libraries for candidate sFRP disrupting molecules are wellknown in the art and, in light of the present disclosure, can now beemployed to identify compounds which disrupt sFRP binding to the peptidemotif (for example SEQ ID NO:9) or TNF family members such as RANKL orTRAIL.

In light of the present disclosure, a variety of affinity bindingtechniques well known in the art can be employed to isolate proteins(i.e. lead compounds) or other compounds. In general, sFRP, a fragmentthereof or the peptide motif (for example a fragment of about three orabout five amino acids of SEQ ID NO: 9) can be immobilized on asubstrate (e.g. a column or filter) and a solution containing a TNFreceptor or a sFRP family member protein can be introduced to the columnto allow formation of the sFRP/TNF or peptide motif/sFRP complex. Then asolution including the test compound(s) is introduced to the columnunder conditions that are permissive for binding. The substrate is thenwashed with a solution to remove unbound or weakly bound molecules. Asecond wash can then elute those compounds that strongly bound to theimmobilized sFRP or peptide motif. Alternatively, the test compounds canbe immobilized and a solution containing sFRP/RANKL or sFRP/peptidemotif (for example SEQ ID NO: 9) can be contacted with the column,filter or other substrate. The ability of either the sFRP or fragmentthereof, or the peptide motif to bind to the test compound can bedetermined as above.

IX. Incorporation of sFRP Therapeutically Effective Fragments, Fusions,and Variants of sFRP or the Peptide Motif into PharmaceuticalCompositions and Methods of Treatment

For administration to animals, purified sFRP, sFRP fragments, sFRPvariants, or peptide motifs that bind sFRP are generally combined with apharmaceutically acceptable carrier. Pharmaceutical preparations cancontain only a single peptide, or can be composed of more than onevariety of sFRP fragments and/or peptide motifs. In general, the natureof the carrier will depend on the particular mode of administrationbeing employed. For instance, parenteral formulations usually compriseinjectable fluids that include pharmaceutically and physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, glycerol, human albumin or the like as avehicle. For solid compositions (e.g. powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

As is known in the art, protein-based pharmaceuticals can be onlyinefficiently delivered through ingestion. However, pill-based forms ofpharmaceutical proteins can alternatively be administeredsubcutaneously, particularly if formulated in a slow-releasecomposition. Slow-release formulations can be produced by combining thetarget protein with a biocompatible matrix, such as cholesterol. Anotherpossible method of administering protein pharmaceuticals is through theuse of mini osmotic pumps. As stated above a biocompatible carrier wouldalso be used in conjunction with this method of delivery.

It is also contemplated that the peptide motifs disclosed herein as wellas sFRP could be delivered to cells in the nucleic acid form andsubsequently translated by the host cell. This could be done, forexample through the use of viral vectors or liposomes. Liposomes couldalso be used for the delivery of the protein itself.

The pharmaceutical compositions of the present disclosure can beadministered by any means that achieve their intended purpose. Amountsand regimens for the administration of sFRP fragments can be determinedreadily by those with ordinary skill in the clinical art of treatingconditions associated with abnormal bone remodeling. For use in treatingthese conditions, the described proteins are administered in an amounteffective to either increase osteoclastogenesis activity or decreaseosteoclastogenesis. Such dosages include amounts which raise targettissue concentrations to levels at which the therapeutic activity hasbeen observed in vitro. The proteins disclosed herein can also be usedto modulate T-cell interactions and immune system functions. Dosessufficient to achieve a tissue concentration that causes an increase ora decrease in osteoclastogenesis and/or T-cell activity can bedetermined by using the amounts described in the examples that follow.The peptides or proteins can be administered to a host in vivo, such asfor example, through systemic administration, such as intravenous orintraperitoneal administration. Also, the peptides or proteins can beadministered intralesionally: i.e. the peptide or protein is injecteddirectly into the tumor or affected area.

Effective doses of the disclosed peptides for therapeutic applicationwill vary depending on the nature and severity of the condition to betreated, the age and condition of the subject and other clinicalfactors. Thus, the final determination of the appropriate treatmentregimen will be made by the clinician. Typically, the dose range will befrom about 0.1 μg/kg body weight to about 100 mg/kg body weight. Othersuitable ranges include doses of from about 1 μg/kg to 10 mg/kg bodyweight. The dosing schedule can vary from once a week to daily dependingon a number of clinical factors, such as the subject's sensitivity tothe protein. Examples of dosing schedules are 3 μg/kg administered twicea week, three times a week or daily; a dose of 7 μg/kg twice a week,three times a week or daily; a dose of 10 μg/kg twice a week, threetimes a week or daily; or a dose of 30 μg/kg twice a week, three times aweek or daily. In the case of a more aggressive disease it can bepreferable to administer doses such as those described above byalternate routes including intravenously or intrathecally. Continuousinfusion can also be appropriate.

EXAMPLES

Polypeptides that bind to sFRP-1 were identified using an open-endedapproach. This approach involved screening a peptide phage display cDNAlibrary for sequences that bound to recombinant sFRP-1 (Uren et al., J.Biol. Chem. 275:4374-4382, 2000). Peptides that had been identified byphage display were operably linked to a sequence encoding alkalinephosphatase creating a fusion protein that, upon binding to sFRP-1,could be detected. This methodology resulted in the identification of apredominant peptide motif containing the sequence L/V-D-G-R-W-L/V (SEQID NO: 9). Alanine scanning was then used to further characterize thepeptide motif (SEQ ID NO: 9 and SEQ ID NOs: 14-26).

The sequence of the peptide motif that binds sFRP was then used toidentify proteins that potentially bind to sFRP-1. The RANKL protein wasidentified as a potential candidate because it contained an amino acidsequence that is similar to that of the peptide motif (SEQ ID NO: 9).ELISA analysis using RANKL and sFRP-1 indicated that sFRP-1 binds toRANKL. RANKL is known to be involved with osteoclast differentiation.Subsequently, sFRP-1 (SEQ ID NO: 3) was shown to inhibitosteoclastogenesis. Moreover, a synthetic peptide containing the peptidemotif enhanced osteoclastogenesis. This finding indicates that thedisruption of the interaction of sFRP-1 (SEQ ID NO: 3) and RANKL withanalogs of the peptide motif (SEQ ID NO: 9) can stimulate osteoclastformation. These materials and methods disclosed herein are exemplaryonly, and are not meant to be limiting.

Example 1 Materials and Methods

1. Materials

Recombinant human sFRP-1 was prepared as described (Uren et al., J.Biol. Chem. 275:4374-4382, 2000). The coding sequence of mouse sFRP-2was amplified by RT-PCR, using total RNA from embryonic mouse kidney asa source, subcloned into pcDNA3.1 expression vector, transfected intoMDCK cells and the recombinant protein purified by heparin-affinitychromatography essentially as described for sFRP-1 in Uren et al., J.Biol. Chem. 275:4374-4382, 2000. Rabbit polyclonal antiserum was raisedagainst recombinant human sFRP-1 by injecting ˜10 μg of purified proteinwith complete Freund's adjuvant into the inguinal lymph nodes, andsubsequently injecting intramuscularly at 2-3 week intervals similarquantities of antigen dissolved in the incomplete Freund's adjuvant.After several boosts, an immunoglobulin fraction was obtained from serumby chromatography with protein G-bound Sepharose (Pharmacia Biotech,Uppsala, Sweden).

Peptides were synthesized using standard solid phase chemistry, purifiedby reverse-phase HPLC and their identity verified by mass spectroscopicanalysis (Research Genetics, Inc., Huntsville, Ala.).

For ELISA assays, recombinant soluble RANKL and TRAIL, and antibodiesdirected against these proteins were obtained from PeproTech (RockyHill, N.J.). Mouse monoclonal antibody (designated anti-FLAG M2)directed against the FLAG epitope was purchased from UpstateBiotechnology, Lake Placid, N.Y. Goat anti-rabbit IgG-alkalinephosphatase and rabbit anti-mouse IgG-alkaline phosphatase conjugatesand paranitrophenolphosphate (pNPP) were purchased from Sigma (St.Louis, Mo.).

For bioassays, recombinant soluble RANKL was purchased from Peprotech,Rocky Hill, N.J., or residues 158-317 of murine RANKL were prepared as aGST-expressed protein. M-CSF was obtained from Research GeneticsInstitute (Boston, Mass., USA).

The M13 phage-displayed random peptide library was constructed asdescribed (Adey et al. Methods in Molecular and Cellular Biology 6:3-14,1995/1996).

Newborn (0-1-day-old) C57BL/6J mice and 6- to 9-week-old male C57BL/6Jmice were purchased from Monash University Animal Services Centre(Clayton, Australia). The murine stromal cell lines, tsJ2, tsJ10 andtsJ14, were generated by transfection with a retroviral vectorexpressing a temperature-sensitive variant of the immortalizing gene ofSV40 (ts A58; Chambers et al., Proc Natl. Acad. Sci. USA 90:5578-5582,1993; Owens et al., Biochem. Biophys. Res. Commun. 222:225-229, 1996).RAW264.7 cells were purchased from the ATCC, and the cell lines KUSA/Oand mc-3T3-el are described in Horwood et al. Endocrinology139:4743-4746, 1998. Osteotropic agents regulate the expression ofosteoclast differentiation factor and osteoprotegerin in osteoblasticstromal cells. 1α,25(OH₂) vitamin D₃ was purchased from Wako PureChemicals Co. (Osaka, Japan). PGE2 was obtained from Sigma (St. Louis,Mo.). Other chemicals and reagents were of analytical grade.

2. Cell Culture

MDCK cells (American Type Culture Collection) were grown in Dulbecco'smodified Eagle's medium (Life Technologies, Inc., Rockville, Md.)containing 10% fetal calf serum (Colorado Serum Company, Denver, Colo.)in 5% CO₂ at 37° C.

3. Screening of Peptide Phage Display Library

Isolation of phage containing sFRP-1-binding peptide segments on theirsurface was performed essentially as previously described (Sparks et al.Screening phage-displayed random peptide libraries. in Phage DisplayPeptides and Proteins Eds. BK Kay et al. Academic Press, NY, 227-253,1996). In brief, a single well in a 96-well ELISA plate (Costar #3590,polystyrene surface) was incubated for 1 hour with purified recombinantsFRP-1 (1 μg/50 μl). This and all other manipulations with ELISA plateswere conducted at room temperature. Subsequently, 150 μl of 1% BSA wasadded to the well and incubated for 2 hours. Following 3 washes withPBS/0.1% Tween 20, 2.5×10¹⁰ phage from the M13 random 12-mer phagedisplay library were added to the pre-coated well and incubated for 3.5hours. After 1 wash with PBS/0.1% Tween 20, the well was incubated for10 minutes with 50 μl of 0.05 M glycine pH 2 to release phage from thesurface. The phage suspension was aspirated from the well, neutralizedwith 50 μl of 0.2 M sodium phosphate, pH 7.4, and amplified for 6-8hours in DH5aF′IQ bacterial broth.

Amplified phage recovered from bacterial broth after this firstenrichment step were subjected to two more rounds of panning in wellscoated with sFRP-1 as described in the previous paragraph, except thatthe phage were incubated for only 2 hours and 1 hour in the second andthird panning steps, respectively. After the third round of panning,phage obtained from the sFRP-1-coated well were titered and seeded on alawn of bacteria to permit isolation of phage from 200 separatecolonies. Bacteria from each of these colonies were grown in broth,pelleted by centrifugation and phage retrieved in the supernatant. Eachof these phage supernatants was tested for binding to sFRP-1-coatedELISA wells versus wells only coated with the BSA blocking solution.Phage were detected in this assay with primary antibody directed againstphage coat protein (Pharmacia Biotech, Uppsala, Sweden, #27-9411-01) andstandard detection reagents. Approximately 100 phage isolates wereselected for sequence analysis, based on exhibiting at least 5-foldhigher binding to sFRP-1 versus BSA coated wells.

4. Sequence Analysis of Peptide Segments Present on the Surface ofIsolated Phage

The sequence of the DNA insert encoding the peptide segment linked tothe M13 gene III coat protein from each phage isolate was determined byusing sequencing primers corresponding to adjacent vector sequence. Anadvanced BLAST search analysis of GenBank databases was performed toidentify proteins that contained sequences matching portions of thepeptide sequences identified by screening of the peptide phage displaylibrary.

5. Generation of Peptide/Alkaline Phosphatase Chimeric Molecules

Synthetic oligonucleotides encoding peptides of interest were ligatedinto the bacterial alkaline phosphatase fusion vector, pMY101, which hadbeen digested with Sal I and Xho I (Yamabhai and Kay, Anal Biochem.247:143-151, 1997). All recombinants were confirmed by DNA sequenceanalysis. Bacteria (E. coli, strain DH5αF′) transformed with thepeptide/AP constructs were grown in Luria broth containing ampicillin(50 μg/mL) to an optical density of 0.5 (at 600 nm), treated with 1 mMisopropyl-β-D-thiogalactopyranoside and then incubated overnight at 37°C. Conditioned medium containing peptide/AP chimera was recovered bycentrifugation at 7000 g for 15 minutes. Chimeric proteins inconditioned medium were stable when stored for a few weeks at 4° C. orfor several months when stored at −80° C.

6. ELISA Analysis of Peptide and Protein Binding to sFRP

ELISA experiments were generally performed as previously described (Urenet al., J. Biol. Chem. 275:4374-4382, 2000), with modificationsdepending on the sFRP binding partner to be tested. Typically, wellswere coated with 0.5 or 1 μg of sFRP-1, blocked with BSA (0.2%, 1%, or4%) and then incubated with putative binding partner overnight at roomtemperature. When investigating the binding of peptide/AP chimeras,after aspiration of bacterial broths, wells were washed and incubatedwith p-nitrophenolphosphate (pNPP). Color development was determined at405 nm with an ELISA reader. For competition experiments, solublepeptides were preincubated with peptide/AP chimeras in bacterial brothfor 30 min at room temperature prior to transfer into ELISA wells coatedwith sFRP-1 or BSA. When testing RANKL binding to sFRP-1, serialdilutions of soluble RANKL were assayed in replicate. Followingovernight incubation at room temperature, RANKL solutions were aspiratedand bound RANKL was detected by sequential incubations with primaryantibody to RANKL, secondary antibody coupled with AP and pNPP. Similarexperimental designs were employed when other TNFα family members wereexamined for binding to sFRPs, and when sFRP-1 derivatives or sFRP-2were the binding targets for RANKL.

7. Isothermal Titration Calorimetry (ITC)

ITC experiments were performed with a VP-ITC MicroCalorimeter (MicroCal,LLC, Northhampton, Mass.) according to the manufacturer's User Manual.In brief, 6 ul aliquots of A-C2 (200 uM, dissolved in PBS) were injectedat regular intervals into a chamber containing sFRP-1 (10 uM, also inPBS). Increases in temperature of the chamber resulting from the bindingof A-C2 and sFRP-1 were determined as a measure of the heat produced bythe binding reaction. Several parameters, included enthalpy anddissociation constant, were calculated from these measurements. Thistechnique is commonly used to quantify the thermodynamic properties ofbinding interactions between proteins and peptides. For instance, seearticle by McNemar et al., Biochemistry 36:10006-10014, 1997.

8. Differential Display PCR

Total cellular RNA was extracted from cell lines or mouse tissues usingguanidine thiocyanate-phenol chloroform and used for reversetranscriptase PCR (RT-PCR) essentially as described (Southby et al.,Endocrinology 137:1349-1357, 1996 and Traianedes et al., J. Biol. Chem.270:20891-20894, 1995). ddPCR was performed essentially as described(Liang et al., Science 257:967-971, 1992 and Traianedes et al., J. Biol.Chem. 270:20891-20894, 1995), except 1 μg of total RNA was reversetranscribed. PCR products were cloned into pCRScriptII (Stratagene,LaJolla, Calif.) or pGEM-T (Promega, Madison, Wis.). DNA sequenceanalysis was performed using a T7 sequencing kit (Pharmacia Biotech,Uppsala, Sweden). Oligonucleotides were synthesized on an Oligo 1000MDNA Synthesizer (Beckman Instruments Inc., Fullerton Calif., USA). Theoligonucleotides were: for ddPCR, DDMR-2 (5′-CTTGATTGCC-3′; SEQ ID NO:37) and T12VA (5-TTTTTTTTTTTT[A,C,G]A; SEQ ID NO: 32-3′).

For ddPCR, the 3′ oligonucleotide is T12VC, where V=A, C, or G. Thisoligonucleotide would anneal to mRNA transcripts having G and B (B=C, G,or T) as the ultimate and penultimate nucleotides prior to the poly Atail. Partial cDNA fragments were amplified using 5′-10 mers resultingin the synthesis of varying length cDNAs due to random annealing todifferent reverse transcribed mRNA species. This PCR reaction isperformed at an annealing temperature of 40° C. and in the presence of[α³⁵S]-dATP to allow the visualization of resulting products. The PCRproducts were resolved on 6% polyacrylamide sequencing gels and exposedto X-ray film for 1-3 days. Differentially regulated cDNA fragments wereexcised from the gel by overlaying the film and cutting out the regionof interest. Using the same oligonucleotides, the cDNA fragment wasreamplified by two rounds of PCR (a total of 80 cycles of PCR). Thereamplified product was then molecularly cloned into pGEM-T (PromegaInc., Madison, Wis.), and the nucleic acid sequence of the amplifiedinsert was determined.

9. sFRP-1 Expression Analysis by RT-PCR

Total RNA isolated from cell lines or tissues was reverse transcribedwith oligo-dT and PCR performed with the primers sfrp-1a(5′-TTAAAATTGCTGCCTGCCTGAG-3′; SEQ ID NO: 38) and sfrp-1b(5′-TCCGAACTACAGGGACAACAGG-3′; SEQ ID NO: 39) for 22 cycles, which wasfound to be in the log-linear phase of amplification for sFRP-1transcripts from osteoblastic sources. Amplifications were performedaccording to manufacturer's instructions. Resultant PCR products wereelectrophoresed, transferred to nylon membrane, and hybridized withα-³²P-labeled internal detection oligonucleotide, sfrp-1c(5′-GCCCAGAGGTATTTCTCAAAGTTG-3′; SEQ ID NO: 39). gapdh-2(5′-ATGAGGTCCACCACCCTGTT-3′; SEQ ID NO: 33, nucleotides 640-659; Tso etal., Nucl. Acids Res. 13:2485-2502, 1985) and gapdh-4 were used toamplify the normalizing gene, glyceraldehyde-3-phosphate dehydrogenase,by 20 cycles of PCR and products were detected with α-³²P-labeledgapdh-1 as described (Suda et al., J. Cell. Physiol. 166:94-104, 1996).

10. SFRP-1 in situ Hybridization Analysis of Tissue Specimens

A murine sFRP-1 riboprobe was generated by PCR using RNA derived fromtsJ2 cells. The resultant fragment of 750 bp was cloned into pGEM-T(Promega, Madison, Wis., USA). The plasmid was linearized andtranscribed with T7 or SP6 RNA polymerase to generate antisense or senseriboprobes. The riboprobes were labeled with digoxigenin (DIG) duringRNA transcription using a RNA labeling kit (Boehringer Mannheim,Mannheim GmbH, Germany) according to the manufacturer's instructions. Insitu hybridization was performed as previously described (Kartsogianniset al., Bone 21:385-392, 1997).

11. Osteoclastogenesis Bioassays

A. Co-culture Systems

Osteoblastic cells were prepared from the calvaria of newborn mice bydigestion with 0.1% collagenase (Worthington Biochemical Co., Freefold,Australia) and 0.2% dispase (Godo Shusei, Tokyo, Japan). Bone marrow andspleen cells were obtained from adult and from newborn mice,respectively (Udagawa et al., J. Exp. Med. 182: 1461-1468, 1995).Osteoblastic cells were co-cultured with bone marrow or spleen cells asdescribed previously (Udagawa et al., J. Exp. Med. 182: 1461-1468,1995). In short, primary osteoblastic cells (2×10⁴/well) and nucleatedspleen cells (1×10⁶/well) or marrow cells (5×10⁵/well) were co-culturedin 48-well plates (Corning Glass Inc., Corning, N.Y.) with 0.4 mL ofα-MEM (GIBCO/BRL, Grand Island, N.Y.) containing 10% fetal bovine serum(Cytosystems, Castle Hill, NSW, Australia) in the presence of testchemicals. Cultures were incubated in quadruplicate and cells werereplenished on day 3 with fresh medium. Osteoclast formation wasevaluated after culturing for 6-7 days. Adherent cells were fixed andstained for tartrate-resistant acid phosphatase (TRAP), and the numberof TRAP-positive osteoclasts was scored as described (Udagawa et al., J.Exp. Med. 182: 1461-1468, 1995). For TRAP staining, adherent cells werefixed with 4% formaldehyde in PBS for 3 minutes. After treatment withethanol-acetone (50/50, vol/vol) for 1 minute, the well surface was airdried and incubated for 10 minutes at room temperature in an acetatebuffer (0.1 M sodium acetate, pH 5.0) containing 0.01% naphthol AS-MXphosphate (Sigma) as a substrate and 0.03% red violet LB salt (Sigma) asa stain for the reaction product in the presence of 50 mM sodiumtartrate. TRAP-positive cells appeared dark red, and those with three ormore nuclei were scored as multinucleated and considered as osteoclasts.Validation of osteoclast formation was achieved using the specificmarker of calcitonin receptor (CTR) expression and bone resorption. CTRexpression was determined either by autoradiography with ¹²⁵I-salmoncalcitonin or by immunohistochemical localization using an array ofantibodies we have developed as described by Quinn et al., Bone 25:1-8,1999.

B. RANKL-induced Osteoclast Formation from Hematopoietic Cells

In some instances, experiments were performed either with adult mousespleen cells or with RAW264.7 cells treated with M-CSF and RANKL asdescribed in Quinn et al., Endocrinology 139:4424-4427, 1998. Whereindicated, these assays were conducted in the presence or absence ofsplenic T cells. T cell fractions were prepared as described in Horwoodet al., Journal of Clinical Investigation 101:595-603, 1998.

Example 2 Identification of Peptides that Bind sFRP-1

To identify peptide sequences that bind sFRP-1, ˜25×10⁹ phages from alibrary containing a diverse repertoire of twelve-amino acid residuesegments linked to the gene III coat protein of M13 phage were screened.After three successive rounds of panning for phage that bound to ELISAwells preincubated with sFRP-1, the phage preparation selected for itsability to bind sFRP-1 was titered and then plated on a lawn ofbacteria. Phage from 200 separate colonies of lysed bacteria werepicked, grown in bacterial broth overnight, recovered in supernatant,and tested for their ability to bind preferentially to sFRP-1 versus BSAin an ELISA. Phage that bound at least five times more avidly to sFRP-1than BSA-coated wells were subjected to nucleotide sequence analysis todetermine the identity of the peptide sequence responsible for thisbinding specificity.

From the approximately 100 phage isolates that were sequenced, elevenunique peptide sequences were deduced. Of note, three of these elevensequences contained a conserved motif consisting of the following sevenamino acid residues: L/V-V-D-G-R-W-L/V (SEQ ID NO: 9). The significanceof this heptapeptide motif was emphasized by the fact that two thirds ofthe phage exhibiting a high specificity for sFRP-1 in the ELISAdisplayed on their surface one of the three sequences with this motif(Table 1).

TABLE 1 SEQ Ref- Amino ID erence Acid Fre- Specificity NO: Code Sequencequency (sFRP-1/BSA) 14 A-C2 QGTLVDGRWLQL 54 10:1 SEQ ID NO: 14 11 A-F4VVDGRWVQGLED  9 10:1 SEQ ID NO: 11 27 B-B9 LVDGRWLYNPHH  4  5:1SEQ ID NO: 27Because of the predominance of this pattern, the binding properties ofthese three peptides, designated A-C2 (SEQ ID NO: 14), A-E4 (SEQ ID NO:1), and B-B9 (SEQ ID NO: 27), and the overall significance of thepeptide motif was further examined. Subsequently, a similar analysis wasperformed with the second mostly frequently observed sequence identifiedby peptide phage display analysis, which was designated A-D9:WECAMYDGRCLT (SEQ ID NO: 40).

Example 3 Confirmation of sFRP-1 Peptide Motif Binding Activity

A set of peptide-alkaline phosphatase fusion proteins containing thepeptide motifs (SEQ ID NOS: 14, 11, and 27) were generated. These fusionproteins were tested for specific binding to sFRP-1 (SEQ ID NO: 3) in anELISA format. As illustrated in FIG. 1, broths from multiple isolates ofthe A-C2 (SEQ ID NO: 14)/alkaline phosphatase fusion protein all showedstrong, highly specific binding to wells preincubated with sFRP-1.Similar results were obtained with the A-E4 (SEQ ID NO: 11)/alkalinephosphatase fusion protein. However, the B-B9 (SEQ ID NO: 27)/alkalinephosphatase fusion protein did not exhibit specific binding to sFRP-1.This qualitative difference between A-C2 (SEQ ID NO: 14), A-E4 (SEQ IDNO: 1), and B-B9 (SEQ ID NO: 27) derivatives was consistent with aquantitative difference noted during the ELISA screening of therespective phage. The A-C2-(SEQ ID NO: 14) and A-E4-(SEQ ID NO: 11)expressing phage were more abundant in the phage preparation selectedfor sFRP-1 binding (Table 1) and showed a higher ratio of sFRP-1:BSAbinding than B-B9 (SEQ ID NO: 27) phage. The more dramatic contrastobserved with the fusion proteins is attributable to the difference invalency of the binding entities: each phage particle has five copies ofthe peptide displayed on its surface, whereas the peptide-alkalinephosphatase fusion proteins exist as dimers in solution. Thus, therelatively weaker binding avidity of the B-B9 sequence as originallyperceived with the pentavalent phage particle became more obvious whendimeric reagents were tested. These results indicate that bindingassociated with the peptide motif could be influenced by the compositionof nearby amino acid residues.

Subsequent experiments demonstrated that the peptide motif (SEQ ID NO:9) was a factor in the binding of the A-C2 (SEQ ID NO: 14)/alkalinephosphatase fusion protein to sFRP-1. For instance, dose-dependentinhibition of A-C2 (SEQ ID NO: 14)/alkaline phosphatase fusion proteinbinding to sFRP-1 was observed with A-C2 (SEQ ID NO: 14) but not with acontrol synthetic peptide (FIG. 2). Alkaline phosphatase itself showedno preferential binding to sFRP-1-coated wells. Individual substitutionsof an alanine residue at each of the twelve sites in the A-C2 sequence(SEQ ID NO: 14) of the A-C2 (SEQ ID NO: 14)/alkaline phosphatase fusionprotein established that all five core residues (V-D-G-R-W) (SEQ ID NO:42) of the native peptide motif enhanced sFRP-1 binding, as did theresidues immediately flanking this core sequence (FIG. 3). Alaninesubstitutions at other sites also had an impact on binding, in somecases increasing the binding avidity for sFRP-1 (SEQ ID NO: 3).

Taken together, peptide phage display analysis followed by ELISAexperiments with peptide/alkaline phosphatase fusion proteinsestablished the existence of a peptide motif (SEQ ID NO: 9) that bindsto sFRP-1. Moreover, binding of proteins containing this peptide motif,L/V-V-D-G-R-W-L/V (SEQ ID NO: 9), could be either enhanced or diminishedby changes in the composition of residues in close proximity to thepeptide motif (SEQ ID NO: 9).

Isothermal titration calorimetry (ITC) was used to demonstrate bindingof AC2 peptide and sFRP-1 in solution, and estimate affinity of theinteraction. Binding was evident, as heat was generated when aliquots ofAC2 solution were added to a chamber containing sFRP-1 dissolved in PBS.By contrast, no heat was produced when AC2 was added to a chambercontaining only PBS. The calculated Kd was 3.9+/−0.46 micromolar (FIGS.4A and 4B).

ELISA experiments were performed essentially as described above with aseries of sFRP-1 deletion mutants (in Uren et al., J. Biol. Chem., 275:4374-4382, 2000) to determine what region(s) of the protein wererequired for binding to the AC2/alkaline phosphatase chimera. Optimalbinding was observed with the Δ3 derivative, which contains all of theFz CRD and a portion of the C-terminal region. Little binding wasdetected with derivatives that contained the CRD alone or the C-terminalregion alone.

Thus, a combination of elements from the CRD and the C-terminal domainwere required for AC2 binding. As derivatives that did not bind well tothe AC2 chimera bound other reagents, and in some instances showedbiological activity, they are unlikely to be simply misfolded.

Another peptide, A-D9, was analyzed in a manner similar to the routinefollowed for A-C2. In particular, ELISA experiments performed with anA-D9/AP chimera showed that this chimera bound specifically to wellscoated with sFRP-1 rather than BSA. This binding was blocked in adose-dependent manner with soluble synthetic peptide containing the A-D9sequence. Binding of the A-D9/AP chimera to sFRP-1 in ELISA wells wasdisrupted by alanine substitutions in the A-D9 sequence. Interestingly,A-C2 peptide also could inhibit binding of the A-D9/AP chimera to sFRP-1and the A-D9 peptide inhibited binding of the A-C2/AP chimera to sFRP-1.This implied that A-C2 and A-D9 recognized overlapping binding sites onsFRP-1, consistent with the presence of a common element (DGR) in thetwo peptides.

Example 4 Identification of Proteins with Sequences Resembling thePeptide Motif

BLAST analysis of sequences in GenBank indicated that the newlydiscovered peptide motif (SEQ ID NO: 9) was not present in any Wntproteins. However, similar sequences were observed in a handful of otherproteins, as illustrated in Table 2.

TABLE 2 Identified Protein with Homology to Peptide SEQ ID NO: MotifAmino Acid Sequence 29 Netrin receptor (UNC5H3) TLCPVDGRW 28 RANKLMVDGSWLDL 10 ANP receptor A (human) VVDGRFVLKITD

The V-D-G-R-W (SEQ ID NO: 42) segment in UNC5H3 was noteworthy becausethis protein is a netrin receptor. Thus, it is possible that sFRP-1 (SEQID NO: 3) interacts with UNC5H3 in a ligand/receptor relationship. Thepresence of the sequence M-V-D-G-S-W-L (SEQ ID NO: 28) inRANKL/TRANCE/OPGL also is notable because of additional evidence thatsFRP-1 (SEQ ID NO: 3) and RANKL are co-expressed in many tissues,including bone where RANKL has a critical role in osteoclast formation(see below). The sequence V-V-D-G-R-F-V (SEQ ID NO: 10) in the humanatrial natriuretic peptide (ANP) receptor A is also of significancebecause of the co-expression of this gene product and sFRP-1 in tissueswithin the kidney and eye. As described herein, sFRP-1 (SEQ ID NO: 3)and RANKL interact with each other in a manner that has significantbiological consequences, and their interaction can be modulated toaffect osteoclastogenesis.

Example 5 sFRP-1 and Expression in Bone

In situ hybridization analyses of sFRP-1 transcripts (SEQ ID NO: 1) inskeletal structures of mouse embryos (Day 19), newborn mice (Day 1) andadult mice (five weeks) were performed to examine the role of sFRP-1 inbone development. Hypertrophic chondrocytes were strongly positive inmurine embryos (E119). In the spinal cord of Day 1 mice, there was verystrong expression in the ossification center within the cartilageprimordium of the lumbar vertebral body and the nucleus pulposus in thecentral part of the lumbar invertebral disc. In the adult, bone liningcells were positive as well as a number of isolated marrow cells, andosteocytes were weakly positive. sFRP-1 mRNA was also observed in theepidermis. RANKL is expressed in a similar pattern (Kartsogiannis etal., Bone 25:525-534, 1999). Expression of sFRP-1 in skeletal sites wasalso detected. Hence, it is likely that sFRP-1 is involved in skeletalmorphogenesis and sFRP-1 expression continues in a number of sitesthrough to adulthood.

sFRP-1 expression in osteoblasts (tsJJ2 cells) was studied (for adescription of the tsJJ2 cell line and the tsJ14 cell line see Chamberset al., Proc. Natl. Acad. Sci. USA 90:5578-5582, 1993). The resultsshowed that sFRP-1 is preferentially expressed in osteoblasts (tsJ2cells) that promote osteoclast formation. Murine sFRP-1 transcripts wereamplified using the oligonucleotides sfrp-1a and sfrp-1b. Amplifiedproducts were verified by Southern analysis using [α-³²P]dATPend-labeled oligonucleotide sfrp-1b as a probe. Differential display PCR(ddPCR) also showed that sFRP-1 is upregulated in osteoblast lines thatstimulate osteoclastogenesis, but not in the products from two otherlines that do not support osteoclast differentiation. Semi-quantitativeRT-PCR analysis of sFRP-1 expression confirmed that transcript level wasmuch higher in lines that were capable of promoting osteoclast formationin co-cultures with hematopoietic progenitor cells. This pattern wasobserved when additional osteoblast lines were compared, reinforcing thefinding that sFRP-1 expression was associated with osteoclastogenesis.

However, in general, osteotropic factors such as 1α,25(OH₂) vitamin D₃caused limited stimulation of sFRP-1 expression by osteoblastic lines.Total RNA was isolated from either untreated or cells treated with1α,25(OH₂) vitamin D₃ for 24 hours, reverse transcribed with oligo (dT),and subjected to PCR for murine SFRP1 and GAPDH. A co-culture ofosteoblasts and bone marrow treated for 24 hours with 1α,25(OH₂) vitaminD₃ was included as a positive control. The primer combination of sfrp-1a(5′-AGC CTT GGC AGT CAA CGA CG-3′ SEQ ID NO: 30) and sfrp-1b (5′-GTT GTGGCT TTT GCA TTG CAC-3′ SEQ ID NO: 31) was used for sFRP-1 amplificationand the primer combination of gapdh-2 (5′-ATG AGG TCC ACC ACC CTG TT-3′SEQ ID NO: 33) and gapdh-4 (5′-CAT GGA GAA GGC TGG GGC TC-3′ SEQ ID NO:34) was used for GAPDH amplification. The resultant PCR products wereelectrophoresed, transferred to nylon membrane and hybridized with[α-³²P]-labeled internal detection oligonucleotide, sfrp-1c (5′-TGT TGAAAA CTA GTA GCT G-3′ SEQ ID NO: 35) and gapdh-1 (5′-GCT GTG GGC AAG GTCATC CC-3′ SEQ ID NO: 36), respectively, as described (Southby et al.,Endocrinology 137:1349-1357, 1996). RT-PCR analysis was repeated intriplicate. Semiquantitative RT-PCR analysis was performed three timeson each RT reaction and two independent RT reactions were examined.

These results indicate that sFRP-1 may be a mediator of hormonallydependent osteoclast formation. On the other hand, sFRP-1 expressionincreased markedly when osteoblasts and osteoclast progenitors wereco-cultured. The time course of this increase matched the rise inappearance of TRAP+ cells, a marker of osteoclast differentiation. Theseresults indicate that upregulation of sFRP-1 expression is dependent oncell-cell communication between the osteoblast and osteoclast lineages.In particular, the correlation between sFRP-1 expression and osteoclastformation suggested that sFRP-1 induction might be a consequence ofosteoclastogenesis.

Example 6 sFRP-1 Blocks Osteoclastogenesis in Cell Culture Bioassays

The possibility that sFRP-1 (SEQ ID NO: 3) and RANKL interact directlywith each other was tested using an ELISA assay. The ELISA assayinvolved the use of wells that were coated with recombinant sFRP-1 (SEQID NO: 3) and subsequently blocked with BSA. RANKL was then incubated inthese wells and in adjacent wells that had only been treated with BSA.Subsequent detection with RANKL antiserum and secondary reagentsrevealed that RANKL bound specifically to sFRP-1 (FIG. 5). This resultwas confirmed in several separate experiments. The use of recombinantreagents indicates that sFRP-1 (SEQ ID NO: 3) and RANKL bind directly toeach other.

The effect of sFRP-1 was assessed upon a RANKL-independent method ofosteoclast formation using the monocyte/macrophage cell line RAW264.7(Quinn et al., Journal of Bone and Mineral Research. 16, 1787-1794,2001) and was compared with that of osteoprotegerin (FIG. 14). In theabsence of TGFα, only limited numbers of osteoclasts are produced formTNFα-treated RAW264.7 cells (Quinn et al., Journal of Bone and MineralResearch. 16, 1787-1794, 2001), so TGFβ was added during the first threedays of culture to increase osteoclast numbers (FIG. 14). sFRP-1inhibited TNFα-dependent osteoclast formation when present during thefirst three days of culture, whilst OPG had no effect suggesting thatsFRP-1 was acting indirectly of RANKL, through binding to TNFα orthrough WNT signaling.

The effect of bacterially expressed CRD was assessed in three differentcell culture models of osteoclast formation. These were: (1) bone marrowcells+RANKL+M-CSF, (2) the macrophage/monocyte cell line RAW264.7+RANKL,and (3) RAW264.7+TNFα+TGFβ (Horwood et al., Journal of Immunology166:4915-4921, 2001; Quinn et al., Journal of Bone and Mineral Research.16, 1787-1794, 2001). In each system, both RANKL-dependent (cultures 1and 2) and RANKL-independent (culture 3, TNFα-dependent osteoclastformation), the bacterially expressed CRD mimicked the action offull-length sFRP-1 and with similar potency (FIG. 15).

As shown above, ELISA experiments with sFRP-1 deletion mutants indicatedthat the Al derivative (Uren et al. J. Biol. Chem., 275: 4374-4382,2000), which consists essentially of the Fz CRD, retained good bindingto RANKL (see FIG. 12). In addition, RANKL binding to a preparation ofbacterially expressed CRD was strong.

Scatchard analysis of the ELISA data indicates that there are twodifferent binding sites: a high affinity site and a low-affinity sitewith affinities of 5-10 nM and 80-120 nM, respectively (see FIG. 13)(see Meshul et al., J. Neurochem. 67:1965, 1996).

Assays measuring the effect of sFRP-1 on osteoclastogenesis showed thatsFRP-1 has a dose-dependent inhibitory activity on osteoclast formation(FIG. 6A). These results were observed in co-cultures of primaryosteoblasts and bone marrow cells treated with vitamin D3 (10⁻⁸ M) andPGE2 (10⁻⁷ M). sFRP-1 reduced the number of multinucleated TRAP+ cellsby 50% when used at a concentration 300 ng/mL, while a dose of 1.6 ug/mLdecreased the number of cells by 95% (FIG. 6A). A similar dose-responsepattern was observed when adult mouse spleen cells were treated withRANKL and M-CSF (FIG. 6B). These results indicate that a directinteraction between sFRP-1 and RANKL blocked osteoclast differentiation.

The significance of data obtained with recombinant sFRP-1 wasstrengthened by the results of experiments performed with proteinG-purified rabbit polyclonal antibodies raised against recombinantsFRP-1. This antibody preparation caused a seven- to ten-fold increasein mononucleated and multinucleated TRAP+ cells in co-cultures ofprimary osteoblasts and adult spleen cells that had been treated withsubmaximal does of D3 (10⁻¹⁰ M) and Dex (110-9 M) (FIG. 7A).Approximately a two to three-fold increase in these cells was observedin co-cultures receiving optimal doses of D3 (10⁻⁸ M) and PGE2 (10⁻⁷ M)(FIG. 1). These results indicated that naturally occurring sFRP-1 waspresent in the cultures and inhibited osteoclast formation. Byneutralizing this endogenous activity, sFRP-1 antibodies boosted thenumber of TRAP+ cells produced in the co-cultures.

Bacterially expressed CRD blocked osteoclast formation in threedifferent cell culture models, mimicking action of full-length sFRP-1and with similar potency (FIG. 14)

The three conditions used in these assays were: (1) bone marrow cells+RANKL+M-CSF, (2) RAW264.7+RANKL, and (3) RAW264.7+TNFα+TGFβ. Withoutbeing bound by theory, as activity was seen in group (3) (in the absenceof RANKL), it is possible that that CRD binds to TNFα, which isstructurally-related to RANKL.

Example 7 A-C2 Synthetic Peptide Promotes Osteoclast Formation

Because the A-C2 (SEQ ID NO: 14) polypeptide has sequence homology toRANKL and because A-C2 (SEQ ID NO: 14) binds to sFRP-1, assays wereperformed to determine if A-C2 (SEQ ID NO: 14) would block sFRP-1binding to RANKL and thus increase osteoclastogenesis. Consistent withthis hypothesis, treatment of osteoblast and adult spleen cellco-cultures with A-C2 (SEQ ID NO: 14) resulted in a ten-fold increase inTRAP+ multinucleated cells (FIG. 8). A-C2 (SEQ ID NO: 14) only had aneffect on osteoclast formation when it was present during day 0 to day 3of the co-culture experiment. RANKL presence is also required forosteoclastogenesis during day 0 to day 3 (Suda et al., Endocrine Reviews20:345-357, 1999). Thus, these results are consistent with the notionthat A-C2 (SEQ ID NO: 14) most likely has an impact on RANKL activity.

Additionally, the positive effect of A-C2 (SEQ ID NO: 14) in theco-culture assays was correlated with the presence of T cells. When Tcells were removed from the adult spleen cell preparations withantibody-coupled magnetic beads, osteoclast formation increased andthere was no additional response to A-C2 (SEQ ID NO: 14; FIG. 10).Similarly, A-C2 (SEQ ID NO: 14) dose-dependent stimulation of TRAP+,multinucleated cell differentiation in RAW264.7 cultures was onlyobserved when T cells were added to the cultures (FIG. 11). Theseresults indicate that T cells express an inhibitory factor (most likelysFRP-1) that is blocked by A-C2 (SEQ ID NO: 14).

Example 8 Structural Analysis Shows that sFRP-1/RANKL Binding ExtendsBeyond the Peptide Motif

Structure-function analysis of sFRP-1/RANKL interaction was performed bytesting RANKL's ability to bind to a set of sFRP-1 deletion mutants(FIG. 12). The strongest binding was observed with a derivative thatcontained the CRD, and strong binding was subsequently seen with asimilar variant comprising the CRD that was expressed in bacteria. Ofnote, we also observed binding of sFRP-1 to a derivative of RANKL thatlacked the sequence corresponding to the A-C2 motif (this RANKL varianthad an amino-terminal sequence beginning with residue 158). This impliedthat the binding of RANKL and sFRP-1 did not rely entirely on thepresence of the A-C2 sequence. Moreover, it implied that other proteinsstructurally related to RANKL but lacking the A-C2-like sequence mightalso bind sFRP-1. We have tested this hypothesis and now have evidencethat TNFα also can bind sFRP-1 in an ELISA format, using conditionscomparable to those described above for RANKL binding studies. Moreover,similar experiments performed with sFRP-2 indicate that it can bind toRANKL and another TNFα family member, the TNF-related apoptosis-inducingligand (TRAIL). Thus, we now believe that additional interactions occurbetween members of the sFRP and TNF families, besides the one involvingsFRP-1 and RANKL.

Example 9 Identification of a Biologically Relevant Peptide Motif

The peptide motif, L/V-V-D-G-R-W-L/V (SEQ ID NO: 9), has not beenpreviously identified as being capable of binding to sFRP-1. Thisbinding activity has been characterized through the use of a series ofELISA experiments using peptide/alkaline phosphatase fusion proteins,alanine scanning mutagenesis and synthetic peptides. These experimentsalso demonstrated that substitutions in nearby residues could enhance orreduce the peptide motif/sFRP-1 binding, implying that systematicsubstitutions in adjacent residues and conservative changes within thepeptide motif, are able to strengthen the interaction with sFRP-1.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Such substitutionsgenerally are conservative when it is desired to finely modulate thecharacteristics of the protein. Examples of amino acids which can besubstituted for an original amino acid in a protein and which areregarded as conservative substitutions include: Ser for Ala; Lys forArg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp forGlu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val forLeu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe;Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile orLeu for Val. The binding affect of peptides in which such substitutionshave been made can readily be confirmed by the peptide motif-bindingassay disclosed herein.

More substantial changes in function or other features can be obtainedby selecting substitutions that are less conservative than thosedescribed above, i.e. selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The effects of these amino acid substitutions or deletionsor additions can be assessed through the use of the ELISA binding assayprovided herein.

Additionally, one of skill in the art will appreciate that nucleic acidsequences encoding the peptide motif can be designed by eithersynthetically synthesizing the appropriate nucleic acid sequence, or byusing PCR to amplify the appropriate sequence. Once obtained the nucleicacid sequence can be placed in an appropriate expression vector andtransformed into an organism such that the organism then produces thepeptide.

The significance of the peptide motif (SEQ ID NO: 9) was demonstrated inexperiments that revealed a dramatic stimulatory effect of a syntheticpeptide bearing the peptide motif in osteoclastogenesis bioassays. Theability of the A-C2 peptide (SEQ ID NO: 14) to promote osteoclastformation was consistent with the idea that the peptide increased RANKLactivity by blocking the inhibitory effect of endogenous sFRP-1 (FIG.17).

The requirement of T cells for the A-C2 peptide (SEQ ID NO: 14) tostimulate osteoclast formation implied that splenic T cells express anosteoclastogenesis inhibitor sensitive to A-C2 (SEQ ID NO: 14). Thisinhibitor is believed to be sFRP-1, as sFRP-1 is expressed in spleen(Finch et al., Proc. Natl. Acad. Sci. USA 94:6770-6775, 1997). Hence,reagents possessing a sFRP-1 binding motif are believed to have utilityin regulating RANKL signaling involved in T cell-dendritic communicationthat is modulated by endogenous sFRP-1. Such regulation can be exploitedto optimize vaccine therapies in a variety of settings where Tcell-dendritic cell interactions have an important role in the immuneresponse.

Dendritic cells have been shown to promote vaccine responses, which canbe determined by measuring titers to known antigens developed ininoculated animals (M. Di Nicola et al., Cytokines Cell. Mol. Ther. 4:265-273, 1998; C. Reis e Sousa et al., Curr. Opin. Immunol. 11: 392-399,1999; K. Tarte and B. Klein, Leukemia 13: 653-663, 1999). Efforts areunderway to optimize the expansion of immunologically responsivedendritic cells in order to improve the efficacy of vaccine therapy (R.Hajek and A. W. Butch, Med. Oncol. 17: 2-15, 2000). Using methods citedin the above references, reagents corresponding to the sFRP-1 bindingmotif and sFRP-1 can be useful to enhance the immune response in vaccinetherapies.

The association of sFRP-1 and T cells indicates that sFRP-1 can beuseful for modulating endogenous proteolytic cleavage of RANKL. A largefraction of the RANKL expressed by T cells is proteolytically processedto release a soluble, biologically active form. This process, which canbe mediated by the TNFα converting enzyme (TACE), involves cleavage atone or two sites in the RANKL sequence just upstream of the putativepeptide-binding motif (SEQ ID NO: 9). Therefore, it is believed thatsFRP-1 regulates the proteolytic processing of RANKL in a manner thatcould be reversed by reagents containing the peptide motif (SEQ ID NO:9).

The ability of sFRP-1 to regulate RANKL processing is tested byculturing T-cells expressing RANKL and treating the cultures withvarious concentrations of sFRP-1. The resulting soluble RANKL proteinsare then identified using a RANKL specific western blot. The degree ofRANKL processing is then correlated to the sFRP concentration in thesample.

In addition to the peptide motif's (SEQ ID NO: 9) impact on sFRP-1/RANKLbinding, reagents containing the peptide motif (SEQ ID NO: 9) also haveutility in disrupting the interaction of sFRP-1 with other proteins. Asmentioned above, other known proteins like the netrin receptor, andUNC5H3 and the ANP receptor A, have sequences similar to the bindingmotif, and newly identified gene sequences can be routinely screened forsuch sequences. Proteins with this motif are likely to be additionalpotential binding partners for sFRP-1 and targets for reagentscontaining the sFRP-1 binding motif. For instance, sFRP-1 binding to theANP receptor A could regulate the release of sodium and fluid in thekidney and eye. Others have demonstrated that the relevant components ofnatriuretic peptide system are functionally expressed in the human eyewhere they could serve as modulators of intraocular pressure (J. Ortegoand M. Coca-Prados, Biochem. Biophys. Res. Commun. 258: 21-28, 1999). Inthe eye, sFRP-1 or its binding peptide could have an important impact onthe release of fluid into the eye with resultant changes in theintraocular pressure. These effects are tested using models of perfusedeyes in organ culture, such as the one originally described by D. H.Johnson and R. C. Tschumper, Invest. Ophthalmol. Vis. Sci. 28: 945-953,1987. Regulation of intraocular pressure has therapeutic benefit in thetreatment of glaucoma.

Example 10 sFRP-1 Blocks Osteoclast Formation

As demonstrated herein, sFRP-1 has an inhibitory effect onosteoclastogenesis, which is likely due to its interaction with RANKL.The elevated expression of sFRP-1 transcript specifically in osteoblastlines capable of stimulating osteoclast formation initially suggestedthat sFRP-1 promotes osteoclastogenesis. However, the increase in sFRP-1transcript observed in co-cultures of osteoblasts and hematopoieticprogenitors as osteoclast formation proceeded implied that sFRP-1 wasinstead part of a tonic mechanism to limit the extent of osteoclastformation (FIG. 17). It is believed that this is important forhomeostasis to ensure that an appropriate balance of osteoblast andosteoclast populations is maintained, along with a reserve of osteoclastprogenitors that would be available when needed in the future. Inaddition, it is possible that low concentrations of sFRP-1 might have apermissive effect on osteoclast formation.

In view of sFRP-1 's ability to inhibit osteoclastogenesis, sFRP-1 canhave clinical utility in conditions where excessive osteoclast activityhas pathological consequences. Osteoporosis and hypercalcemicosteopaenia are examples of such conditions; rheumatoid arthritis isanother, which could be a particularly good target for sFRP-1 therapybecause soluble RANKL from T cells is thought to have an important rolein the bone loss associated with this disease.

Example 11 Other Interactions Between Members of the sFRP and TNFαFamilies

The results described above (Example 8) indicate that additional contactpoints within RANKL, besides the newly identified peptide-binding motif,are involved in the interaction of sFRP-1 (SEQ ID NO: 3) and RANKL.These results further indicate that sFRP family members interact withTNF-ligand family members.

TNF-ligand family members are known to be among the most pleiotropiccytokines, inducing a large number of cellular responses, includingcytotoxicity, anti-viral activity, immunoregulatory activities, and thetranscriptional regulation of several genes. Cellular responses toTNF-family ligands include not only normal physiological responses, butalso diseases associated with increased apoptosis or the inhibition ofapoptosis. Apoptosis-programmed cell death is a physiological mechanisminvolved in the deletion of peripheral T lymphocytes of the immunesystem, and its dysregulation can lead to a number of differentpathogenic processes. Diseases associated with increased cell survival,or the inhibition of apoptosis, include cancers, autoimmune disorders,viral infections, inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. Diseases associated withincreased apoptosis include AIDS, neurodegenerative disorders,myelodysplastic syndromes, ischemic injury, toxin-induced liver disease,septic shock cachexia and anorexia.

Thus, the disclosure further provides methods for modulating the TNFligand/TNF receptor interactions. These methods involve contacting sFRP,a fragment or variant of sFRP, or the peptide motif (SEQ ID NO: 9) witha member of the TNF-ligand family of proteins, and detecting a change inTNF-ligand biological activity.

Whether the sFRP, fragment or variant of sFRP, or the peptide motif actsas an “agonist” or antagonist” can readily be determined using any oneof the well known TNF-family ligand/receptor cellular response assays,such as ones described in the references cited in the following reviews:D. Wallach et al., Annu. Rev. Immunol. 17: 331-367, 1999; S. J. Bakerand E. P. Reddy, Oncogene 17: 3261-3270, 1998

Thus, the disclosure provides screening methods for determining whethera candidate agonist or antagonist is capable of enhancing or inhibitinga cellular response to a TNF-family ligand.

Example 12 sFRP-1/Peptide Binding

ELISA experiments were performed essentially as described above with aseries of sFRP-1 deletion mutants (in Uren et al., J. Biol. Chem., 275:4374-4382, 2000) to determine what region(s) of the protein wererequired for binding to the AC2/alkaline phosphatase chimera. Optimalbinding was observed with the Δ3 derivative, which contains all of theFz CRD and a portion of the C-terminal region. Little binding wasdetected with derivatives that contained the CRD alone or the C-terminalregion alone.

Thus, a combination of elements from the CRD and the C-terminal domainwere required for AC2 binding. As derivatives that did not bind well tothe AC2 chimera bound other reagents, and in some instances showedbiological activity; therefore, they are unlikely to be simplymisfolded.

Having illustrated and described the principles of the disclosure inmultiple embodiments and examples, it should be apparent to thoseskilled in the art that the disclosure can be modified in arrangementand detail without departing from such principles. We claim allmodifications coming within the spirit and scope of the followingclaims.

1. A method of enhancing osteoclast differentiation, comprising:contacting an osteoclast progenitor cell with a purified polypeptide,wherein the polypeptide is less than 30 amino acids in length andcomprises a peptide motif that: (a) binds sFRP-1; and (b) comprises theamino acid sequence as set forth as SEQ ID NO: 9, wherein contacting theosteoclast progenitor cell with the purified polypeptide comprisesselecting a subject with a disorder characterized by increased boneformation, wherein the subject has an insufficient number of osteoclastsor has osteoclasts with decreased activity, and administering thepurified polypeptide to the subject in an amount sufficient to enhanceosteoclast differentiation and remodel bones in the subject.
 2. Themethod of claim 1, wherein the peptide is less than 20 amino acids inlength.
 3. The method of claim 1, wherein the subject has osteopetrosischaracterized by an insufficient number of osteoclasts.
 4. The method ofclaim 3, wherein the peptide is administered in a pharmaceuticalcomposition.
 5. The method of claim 1, wherein the polypeptide comprisesthe formula[R1]_(x)-R2-R3-R4-R5-R6-R7-R8-[R9]_(y), wherein: x and y are integersindependently selected from the group 0 or 1; R1 and R9 comprise anyamino acid residue; R2 is Leu or Val; R3 is Val; R4 is Asp; R5 is Gly;R6 is Arg; R7 is Trp; R8 is Leu or Val.
 6. The method of claim 5,wherein R1 is (a) Gln-Gly-Thr, (b) Ala-Gly-Thr, or (c) Gln-Gly-Ala. 7.The method of claim 5, wherein R9 is (a) Gln-Leu, (b) Ala-Leu, (c)Gln-Ala or (d) Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43).
 8. The method ofclaim 5, wherein R1 is (a) Gln-Gly-Thr, (b) Ala-Gly-Thr, or (c)Gln-Gly-Ala; R2 is Leu, or Val; R8 is Leu or Val; R9 is (a) Gln-Leu, (b)Ala-Leu, (c) Gln-Ala, or (d) Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43); and R3is Val, R4 is Asp, R5 is Gly, R6 is Arg, and R7 is Trp.
 9. The method ofclaim 8, wherein R9 is (a) Gln-Leu, (b) Ala-Leu, (c) Gln-Ala, or (d)Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43).
 10. The method of claim 5, whereinthe polypeptide comprises the amino acid sequence as set forth as SEQ IDNOs: 11, 14-17, 25, or
 26. 11. The method of claim 10, wherein thepeptide comprises the amino acid sequence as set forth as SEQ ID NO: 14.12. The method of claim 11, wherein the peptide consists of the aminoacid sequence as set forth as SEQ ID NO:
 14. 13. The method of claim 1,wherein the polypeptide further comprises a peptide tag, a detectablelabel, or an epitope tag.
 14. The method of claim 1, wherein thepolypeptide is a first amino acid sequence and further comprises asecond amino acid sequence.
 15. The method of claim 14, wherein thefirst amino acid sequence comprises SEQ ID NO:
 9. 16. The method ofclaim 15, wherein the first amino acid sequence comprises SEQ ID NO: 14.17. The method of claim 15, wherein the second amino acid sequencecomprises alkaline phosphatase.
 18. The method of claim 14, wherein thesecond amino acid sequence is not found joined to the peptide in nature.19. The method of claim 14, wherein the first amino acid sequence is afirst domain of a fusion protein and the second amino acid sequence is asecond domain of the fusion protein.
 20. A method of enhancingosteoclast differentiation, comprising: contacting an osteoclastprogenitor cell in vitro with a purified polypeptide, wherein thepurified polypeptide is less than 30 amino acids in length and comprisesa peptide motif that: (a) binds sFRP1; and (b) comprises the amino acidsequence as set forth as SEQ ID NO:
 9. 21. The method of claim 20,wherein the purified polypeptide comprises the formula[R1]_(x)-R2-R3-R4-R5-R6-R7-R8-[R9]_(y), wherein: x and y are integersindependently selected from the group 0 or 1; R1 and R9 comprise anyamino acid residue; R2 is Leu or Val; R3 is Val; R4 is Asp; R5 is Gly;R6 is Arg; R7 is Trp; R8 is Leu or Val.
 22. The method of claim 21,wherein R1 is (a) Gln-Gly-Thr, (b) Ala-Gly-Thr, or (c) Gln-Gly-Ala. 23.The method of claim 21, wherein R9 is (a) Gln-Leu, (b) Ala-Leu, (c)Gln-Ala or (d) Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43).
 24. The method ofclaim 21, wherein R1 is (a) Gln-Gly-Thr, (b) Ala-Gly-Thr, or (c)Gln-Gly-Ala; R2 is Leu, or Val; R8 is Leu or Val; R9 is (a) Gln-Leu, (b)Ala-Leu, (c) Gln-Ala, or (d) Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43); and R3is Val, R4 is Asp, R5 is Gly, R6 is Arg, and R7 is Trp.
 25. The methodof claim 24, wherein R9 is (a) Gln-Leu, (b) Ala-Leu, (c) Gln-Ala, or (d)Gln-Gly-Leu-Glu-Asp (SEQ ID NO: 43).
 26. The method of claim 21, whereinthe polypeptide comprises the amino acid sequence as set forth as SEQ IDNOs: 11, 14-17, 25, or
 26. 27. The method of claim 20, wherein thepolypeptide further comprises a peptide tag, a detectable label, or anepitope tag.